JP2016537346A - Inhibitors of influenza virus replication - Google Patents

Abstract

The polymorphic compound (1) or a pharmaceutically acceptable salt thereof (wherein the compound (1) is represented by the following structural formula) is represented by the form A of the compound (1) · 1 / 2H 2 O HCl: Salt, HCl salt of Form (1) 3H2O, HCl salt of Form D (1), Form A (1) and Form A (1) tosylate salt. Such polymorphs are used to treat influenza, inhibit influenza virus replication, or reduce the amount of influenza virus in a biological sample or subject.

Description

Cross Reference to Related Applications This PCT application claims the benefit of US Provisional Application No. 61 / 903,572, filed November 13, 2013. This document is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION The present invention relates to inhibiting influenza virus replication, treating or reducing the severity of influenza infection in patients, and prophylactically preventing or reducing the incidence of influenza infection in patients. It relates to compounds useful for the preparation and compounds in solid form.

Background Influenza spreads around the world as a seasonal epidemic, which kills hundreds of thousands of people each year and millions in a global epidemic year. For example, the influenza pandemic occurred three times in the 20th century, resulting in the death of tens of millions, and each of these pandemics was caused by the emergence of new strains of the virus in humans It was. These new strains are often due to the transmission of existing influenza viruses from other animal species to humans.

  Influenza is transmitted from person to person primarily through large droplets containing the virus that are produced when the infected person coughs or sneezes; these large droplets are then close to the infected person (e.g. Can stay on the mucosal surface of the upper respiratory tract of susceptible individuals (within about 6 feet). Transmission can also occur through direct or indirect contact with respiratory secretions (eg, touching an eye, nose or mouth after touching a surface contaminated with influenza virus). Adults may be able to transmit influenza to others from one day before symptoms appear until about five days after symptoms begin to appear. Younger children and those with weakened immune systems can be infectious for a period of 10 days or more after the onset of symptoms.

  Influenza viruses are RNA viruses of the Orthomyxoviridae family that includes five genera: influenza virus A, influenza virus B, influenza virus C, ISA virus and Togotovirus.

  The influenza virus A genus has one species, the influenza A virus. For various influenza A, wild waterfowl are natural hosts. Occasionally, the virus can be transmitted to other species and cause a catastrophic pandemic in domesticated poultry, or a global pandemic of human influenza. These type A viruses are the most pathogenic human pathogens of the three influenza types and cause the most severe diseases. Influenza A viruses can be subdivided into different serotypes based on the antibody response to these viruses. The serotypes identified in humans are in the order of deaths due to known global pandemics: H1N1 (cause of Spanish cold in 1918), H2N2 (cause of Asian cold in 1957), H3N2 (Hong Kong in 1968) Causes of cold), H5N1 (a threat of the pandemic in the 2007-08 influenza season), H7N7 (having rare zoonotic potential), H1N2 (specific to humans and pigs), H9N2, H7N2, H7N3 and H10N7 It is.

  Influenza virus B has one species, the influenza B virus. Influenza B almost exclusively infects humans and is less common than influenza A. The only other animal known to be susceptible to influenza B infection is the seal. This type of influenza mutates 2-3 times lower than type A, and as a result, is less genetically diverse and there is only one influenza B serotype. As a result of this lack of antigenic diversity, some immunity against influenza B is usually acquired at a young age. However, influenza B mutates sufficiently that there is no persistent immunity. This low rate of antigen change, coupled with its limited host range (inhibits cross-species antigen shifts), prevents the influenza B pandemic from occurring.

  The influenza virus C genus has one species, influenza C virus, which can infect humans and pigs, causing severe illness and regional epidemics. However, influenza C is less common than the other types and usually appears to cause mild disease in children.

  Influenza A, B and C viruses are very similar in structure. The virus particles are 80-120 nanometers in diameter and are usually nearly spherical, but can also form fibrous forms. Unusually as a virus, its genome is not a single piece of nucleic acid; instead, it contains 7 or 8 segments of negative-sense RNA. The influenza A genome contains 11 proteins: hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), M1, M2, NS1, NS2 (NEP), PA, PB1, PB1-F2 and PB2. Code.

  HA and NA are large glycoproteins on the outer surface of the viral particle. HA is a lectin that mediates virus and target cell binding and viral genome entry into the target cell, and NA is from the infected cell by cleaving the sugar that binds to mature virus particles. Involved in the release of progeny virus. These proteins are therefore targets for antiviral drugs. Furthermore, these are antigens that can produce antibodies. Influenza A viruses are categorized into subtypes based on antibody responses to HA and NA, for example, in H5N1, which provides a basis for H and N discrimination (see above).

  Influenza creates direct costs due to lost productivity and associated medical treatment, as well as indirect costs of preventive measures. In the United States, it is estimated that the future pandemic can cause direct and indirect costs of hundreds of billions of dollars while influenza is responsible for total costs of over $ 10 billion per year. Preventive costs are also high. Countries around the world are preparing for a potential H5N1 avian influenza pandemic, along with the costs associated with the purchase of drugs and vaccines, and the development of strategies for disaster drills and improved border regulations. , Spending billions of dollars to plan.

  Current treatment options for influenza include vaccination and chemotherapy or chemoprevention with antiviral drugs. Vaccination against influenza with influenza vaccines is often recommended for high-risk groups such as children and the elderly, or in people with asthma, diabetes or heart disease. However, even vaccination can cause influenza. Vaccines are recombined against several specific strains of influenza each season, but probably not all strains that actively infect people around the world during that season. It can take six months for a manufacturer to formulate and produce the millions needed to deal with a seasonal epidemic; occasionally, during that time, new or overlooked stocks Notably, people get infected despite being vaccinated (like the H3N2 Fujian cold in the 2003-2004 flu season). Infections can occur shortly before vaccination, and vaccines can take weeks to become effective, so it can be the exact strain that the vaccine is supposed to prevent.

  Furthermore, the effectiveness of these influenza vaccines varies. Due to the high mutation rate of the virus, certain influenza vaccines usually protect only for a few years. Because influenza viruses change quickly over time and different strains predominate, vaccines formulated over a year may be ineffective the following year.

  Also, since there is no RNA proofing enzyme, the RNA-dependent RNA polymerase of influenza vRNA causes one nucleotide insertion error for every approximately 10,000 nucleotides, which is the approximate length of influenza vRNA. Thus, almost all newly created influenza viruses are mutants, ie antigenic drifts. If more than one virus lineage infects a single cell, the genome is separated into 8 separate segments of vRNA, allowing vRNA to be mixed or reassembled. The drastic change in the genetic nature of the resulting virus results in an antigen shift that allows the virus to infect new host species and immediately overcome protective immunity.

  Antiviral drugs can also be used to treat influenza, particularly neuraminidase inhibitors are effective, but viruses can be resistant to standard antiviral drugs. Such agents can be prepared to have a variety of different chemical forms, including chemical derivatives or salts, or have a variety of physical forms. For example, they can be amorphous, have various crystal polymorphs, or exist in various solvated or hydrated states. By changing the form, it may be possible to change its physical properties. Such different forms may have different properties, especially as an oral formulation. Specifically, exhibit improved properties (eg, improved water solubility and stability, better processability or preparation of pharmaceutical formulations, and increased bioavailability of orally administered compositions) It may be desirable to identify improved forms. Such improved properties discussed above can be altered to be beneficial for a particular therapeutic effect.

  Changing the form of the antiviral agent can be one of many ways to adjust the physical properties of such antiviral agents to be more useful in the treatment of influenza.

SUMMARY OF THE INVENTION The present invention generally comprises a polymorphic compound (1) or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable formulation thereof, a compound (1) of such polymorphic form. The method of preparation and the use of such polymorphs to inhibit influenza virus replication, to reduce the amount of influenza virus and to treat influenza.

によって表され、ここで、その多形体は、Ａ形の化合物（１）・１／２Ｈ_２ＯのＨＣｌ塩、Ｆ形の化合物（１）・３Ｈ_２ＯのＨＣｌ塩、Ｄ形の化合物（１）のＨＣｌ塩、Ａ形の化合物（１）およびＡ形の化合物（１）のトシレート塩からなる群より選択される。 In one embodiment, the invention relates to polymorphic compound (1) or a pharmaceutically acceptable salt thereof, wherein compound (1) has the structural formula:

Wherein the polymorphs are represented by Form A compound (1), HCl salt of 1 / 2H ₂ O, Form F compound (1), HCl salt of 3H ₂ O, Form D compound (1 ) HCl salt, Form A compound (1) and Tosylate salt of Form A compound (1).

  In another embodiment, the present invention provides a polymorphic compound (1) disclosed herein or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier or excipient. It relates to a pharmaceutically acceptable formulation comprising.

  In yet another embodiment, the invention relates to a method of inhibiting influenza virus replication in a biological in vitro sample or patient. The method comprises administering to the sample an effective amount of a polymorphic compound (1) disclosed herein or a pharmaceutically acceptable salt thereof.

  In yet another embodiment, the present invention relates to a method for reducing the amount of influenza virus in a biological in vitro sample or patient. The method comprises administering to the sample an effective amount of a polymorphic compound (1) disclosed herein or a pharmaceutically acceptable salt thereof.

  In yet another embodiment, the invention relates to a method of treating influenza in a patient. The method comprises administering to the sample an effective amount of a polymorphic compound (1) disclosed herein or a pharmaceutically acceptable salt thereof.

In yet another embodiment, the present invention relates to a method of preparing HCl salt of Form A compound (1) .1 / 2H ₂ O. The method comprises mixing HCl with compound (1) in a solvent system comprising water and one or more organic solvents, wherein the solvent system has a water activity of 0.05 to 0.85. Have Compound (1) may be solvated or unsolvated and / or may be amorphous or crystalline.

In yet another embodiment, the present invention relates to a method for preparing the HCl salt of compound (1) · 3H ₂ O in Form F. The method comprises the step of mixing HCl and compound (1) in a solvent system comprising water or comprising water and one or more organic solvents, wherein the solvent system is equal to 0.9 or A water activity greater than that, for example having a water activity of 0.9 to 1.0); or a compound of form A (1) in a solvent system comprising water or comprising water and one or more organic solvents Stirring the HCl salt of 1 / 2H ₂ O, where the solvent system has a water activity equal to or greater than 0.9, eg, a water activity of 0.9-1.0 including. Compound (1) may be solvated or unsolvated and / or may be amorphous or crystalline.

In yet another embodiment, the invention relates to a method for preparing the HCl salt of compound (1) in Form D. The method includes the step of dehydrating the HCl salt of Form A compound (1) .1 / 2H ₂ O.

  In yet another embodiment, the invention relates to a method of preparing Form A compound (1). The method comprises the step of stirring the amorphous compound (1) or solvate of compound (1) in a solvent system comprising water and ethanol.

  In yet another embodiment, the invention relates to a method for preparing the tosylate salt of Form A compound (1). The method comprises the step of stirring a mixture of a solvent system comprising amorphous compound (1) or a solvate of compound (1), p-toluenesulfonic acid, and acetonitrile.

  The 2-methyl THF solvate of compound (1) is also encompassed in the present invention.

  In yet another embodiment, the invention relates to a method of reducing the amount of influenza virus in a biological in vitro sample or subject, wherein the method comprises a multiplicity disclosed herein in an effective amount. Administering a form of Compound (1).

  In yet another embodiment, the present invention relates to a method of inhibiting replication of influenza virus in a biological in vitro sample or subject, the method comprising a multiplicity disclosed herein in an effective amount in that sample. Administering a form of Compound (1).

  In yet another embodiment, the invention relates to a method of treating influenza in a subject, the method comprising administering to the subject a therapeutically effective amount of a polymorphic compound (1) disclosed herein. Administering.

  The present invention provides a polymorphic compound (1) disclosed herein for inhibiting influenza virus replication, reducing the amount of influenza virus, or treating influenza in a subject. Includes use. The present invention relates to polymorphs disclosed herein for inhibiting influenza virus replication, reducing the amount of influenza virus, or manufacturing a medicament for treating influenza in a subject. Use of the compound (1).

In yet another embodiment, the present invention provides compound (1) or a pharmaceutically acceptable salt thereof (for example, compound A (1) · 1 / 2H ₂ ) in the range of 100 mg to 1,600 mg. HCl salt of O, Form F compound (1). HCl salt of 3H ₂ O, Form D compound (1) HCl salt, Form A compound (1) and Form A compound (1) tosylate salt) Administration regimen.

1 and 2 are a powder X-ray diffraction (XRPD) pattern and a C ¹³ solid state nuclear magnetic spectroscopy (C ¹³ SS NMR) spectrum of the HCl salt of Form A compound (1) · 1 / 2H ₂ O, respectively. 1 and 2 are a powder X-ray diffraction (XRPD) pattern and a C ¹³ solid state nuclear magnetic spectroscopy (C ¹³ SS NMR) spectrum of the HCl salt of Form A compound (1) · 1 / 2H ₂ O, respectively.

3 and 4 are the XRPD pattern and C ¹³ SSNMR spectrum of the HCl salt of Form F compound (1) · 3H ₂ O, respectively. 3 and 4 are the XRPD pattern and C ¹³ SSNMR spectrum of the HCl salt of Form F compound (1) · 3H ₂ O, respectively.

FIGS. 5 and 6 are the XRPD pattern and C ¹³ SSNMR spectrum of the HCl salt of Form D compound (1), respectively. FIGS. 5 and 6 are the XRPD pattern and C ¹³ SSNMR spectrum of the HCl salt of Form D compound (1), respectively.

FIGS. 7 and 8 are the XRPD pattern and C ^{1 3} SSNMR spectrum, respectively, of Form A compound (1). FIGS. 7 and 8 are the XRPD pattern and C ^{1 3} SSNMR spectrum, respectively, of Form A compound (1).

FIG. 9 is an XRPD pattern of the tosylate salt of Form A compound (1).

FIG. 10 is an XRPD pattern of 2-methyltetrahydrofuran (2-MeTHF) solvate of compound (1).

FIG. 11 is an XRPD pattern of amorphous compound (1).

FIG. 12 is a temperature phase diagram for water activity versus transition between various polymorphs of the HCl salt of compound (1).

FIG. 13 is a graph showing AUC virus shedding for a 1200 mg / 600 mg dose group of Form A compound (1) · 1 / 2H ₂ O HCl salt in a live attenuated influenza challenge model in humans.

Detailed Description of the Invention Solid form

によって表される化合物（１）およびその薬学的に許容され得る塩は、インフルエンザウイルスの複製を阻害し得、ＷＯ２０１０／１４８１９７にも記載されている。 The following structural formula:

Compound (1) and its pharmaceutically acceptable salts represented by can inhibit influenza virus replication and are also described in WO2010 / 148197.

  Compound (1) may exist in various polymorphic forms or may form various polymorphs. As is known in the art, polymorphism is the ability of a compound to crystallize as more than one different crystalline or “polymorphic” species. A polymorph is a solid crystalline phase of a compound in which there are at least two different configurations or polymorphs of the compound molecule in the solid state. Polymorphs of any given compound are defined by the same chemical formula or composition, but differ in chemical structure to the same extent as the crystal structures of two different chemical compounds. In general, different polymorphs can be identified by analytical methods such as powder X-ray diffraction (XRPD) patterns, thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC), or by their melting points, or other techniques known in the art. Can be characterized by As used herein, the term “polymorph” includes solvates and neat polymorphs that do not have any solvate.

  As used herein, “compound (1)” means compound (1) in the form of the free base. Accordingly, “HCl salt of compound (1)” means the HCl salt of the free base compound, and “tosylate salt of compound (1)” means the tosylate salt of the free base compound. Note that unless otherwise specified, compound (1) and the salt of compound (1) may be solvated or unsolvated. It is also noted that compound (1) and the salt of compound (1) may be crystalline or amorphous unless specified otherwise.

In one embodiment, the invention relates to the polymorphic Form A compound (1). HCl salt of 1 / 2H ₂ O. This form is a polymorph of the HCl salt of Compound (1) containing half equivalent water as a solvate per Compound (1). In one specific embodiment, the HCl salt of Form A compound (1) .1 / 2H ₂ O is 10.5, 5.2, 7.4 and 18.9 degrees in the powder X-ray diffraction pattern. Characterized by one or more peaks corresponding to 2-theta values measured in units of (± 0.2 degrees). In another specific embodiment, the HCl salt of Form A compound (1) .1 / 2H ₂ O is 25.2 ± 0.2, 16.5 ± 0.2, in the powder X-ray diffraction pattern, Further characterized by one or more peaks corresponding to 2-theta values measured in units of 18.1 ± 0.2 and 23.0 ± 0.2 degrees. In another specific embodiment, the HCl salt of Form A compound (1) .1 / 2H ₂ O is characterized by 2-theta ± 0.2 at the positions listed in Table 2 below: It is characterized as having an XRPD pattern with a distinct peak. In yet another specific embodiment, the HCl salt of Form A compound (1) .1 / 2H ₂ O is characterized as having substantially the same XRPD pattern as shown in FIG. Their XRPD patterns are obtained at room temperature using CuK alpha radiation. In yet another specific embodiment, the HCl salt of polymorphic Form A compound (1) .1 / 2H ₂ O is 29.2, 107.0, 114.0 and 150 in the C ¹³ SSNMR spectrum. .7 (± 0.3 ppm) is characterized as having one or more characteristic peaks. In yet another specific embodiment, the HCl salt of polymorphic Form A compound (1) .1 / 2H ₂ O is 22.1, 24.6, 47.7 and 54 in the C ¹³ SSNMR spectrum. Further characterized as having one or more characteristic peaks at .8 (± 0.3 ppm). In yet another specific embodiment, the HCl salt of Form A compound (1) .1 / 2H ₂ O is characterized as having a C ¹³ SSNMR peak listed in Table 3. In yet another specific embodiment, the HCl salt of Form A compound (1) .1 / 2H ₂ O is characterized as having substantially the same C ¹³ SSNMR spectrum as shown in FIG. .

In one embodiment, the invention relates to the HCl salt of Compound (1) .3H ₂ O in polymorphic Form F. This form is a polymorph of the HCl salt of compound (1) containing 3 equivalents of water as solvate per compound (1). In one specific embodiment, the HCl salt of Form F compound (1) .3H ₂ O has 7.1, 11.9, 19.2 and 12.4 (± 0) in the powder X-ray diffraction pattern. .2) Characterized by one or more peaks corresponding to 2-theta values measured in degrees. In another specific embodiment, the HCl salt of Form F compound (1) .3H ₂ O is 16.4, 21.8 and 23.9 (± 0.2) degrees in the powder X-ray diffraction pattern. Further characterized by one or more peaks corresponding to 2-theta values measured in units of. In another specific embodiment, the HCl salt of Form F compound (1) .3H ₂ O has a characteristic peak represented by 2-theta ± 0.2 at the positions listed in Table 5 below: It is characterized as having an XRPD pattern with In yet another specific embodiment, the HCl salt of Form F compound (1) .3H ₂ O is characterized as having substantially the same XRPD pattern as shown in FIG. Their XRPD patterns are obtained at room temperature using CuK alpha radiation. In yet another specific embodiment, the HCl salt of polymorphic Form F compound (1) .3H ₂ O is 20.7, 27.4, 104.8, 142.5 in the C ¹³ SSNMR spectrum. Characterized by a peak at 178.6 (± 0.3 ppm). In yet another specific embodiment, the HCl salt of polymorphic Form F compound (1) .3H ₂ O is 154.3, 20.3, 132.3 and 21.1 in the C ¹³ SSNMR spectrum. It is further characterized by one or more peaks corresponding to (± 0.3 ppm). In yet another specific embodiment, the HCl salt of Form F compound (1) .3H ₂ O is characterized as having a C ¹³ SSNMR peak listed in Table 6. In yet another specific embodiment, the HCl salt of Form F compound (1) .3H ₂ O is characterized as having substantially the same C ¹³ SSNMR spectrum as shown in FIG.

In one embodiment, the invention relates to the HCl salt of compound (1) in polymorph D form. This form is the unsolvated form of the HCl salt of compound (1). In one specific embodiment, the HCl salt of Form D compound (1) is measured in units of 5.8, 17.1, and 19.5 (± 0.2) degrees in the powder X-ray diffraction pattern. Characterized by one or more peaks corresponding to the 2-theta values made. In another specific embodiment, the HCl salt of Form D compound (1) is measured in units of 5.3, 10.5, and 15.9 (± 0.2) degrees in a powder X-ray diffraction pattern. Characterized by one or more peaks corresponding to the 2-theta values made. In another specific embodiment, the HCl salt of Form D compound (1) has an XRPD pattern with a characteristic peak represented by 2-theta ± 0.2 at the positions listed in Table 7. Then it is characterized. In yet another specific embodiment, the HCl salt of Form D compound (1) is characterized as having substantially the same XRPD pattern as shown in FIG. Their XRPD patterns are obtained at room temperature using CuK alpha radiation. In yet another specific embodiment, HCl salt of D-type compound ^(1), in the C 13 SSNMR spectrum, 29.4,53.4,113.3,135.4,177.8 (± 0.3ppm) with a peak. In yet another specific embodiment, the HCl salt of Form D compound (1) is 22.9, 23.9, 26.0 and 31.6 (± 0.3 ppm) in the C ¹³ SSNMR spectrum. Is further characterized by one or more peaks corresponding to. In yet another specific embodiment, the HCl salt of Form D compound (1) is characterized as having the C ¹³ SSNMR peaks listed in Table 8. In yet another specific embodiment, the HCl salt of Form D compound (1) is characterized as having substantially the same C ¹³ SSNMR spectrum as shown in FIG.

In one embodiment, the invention relates to polymorph Form A compound (1). This form is the unsolvated free base form of Compound (1). In one specific embodiment, Form A compound (1) is measured in units of 15.5, 18.9 and 22.0 (± 0.2) degrees in a powder X-ray diffraction pattern 2 -Characterized by one or more peaks corresponding to theta values. In another specific embodiment, Form A compound (1) is in units of 11.8, 16.9, 25.5 and 9.1 (± 0.2) degrees in the powder X-ray diffraction pattern. It is further characterized by one or more peaks corresponding to the measured 2-theta values. In another specific embodiment, Form A compound (1) is characterized as having an XRPD pattern with a characteristic peak represented by 2-theta ± 0.2 at the positions listed in Table 10. It is done. In yet another specific embodiment, Form A compound (1) is characterized as having substantially the same XRPD pattern as shown in FIG. Their XRPD patterns are obtained at room temperature using CuK alpha radiation. In yet another specific embodiment, A form of Compound ^(1), in the C 13 SSNMR spectrum, 21.0,28.5,50.4,120.8,138.5 and 176.2 ( It is characterized by having a peak at ± 0.3 ppm). In yet another specific embodiment, Form A compound (1) peaks at 30.1, 25.9, 22.8, and 25.0 (± 0.3 ppm) in the C ¹³ SSNMR spectrum. Characterized as having. In yet another specific embodiment, Form A compound (1) is characterized as having a C ¹³ SSNMR peak listed in Table 11. In yet another specific embodiment, Form A compound (1) is characterized as having substantially the same C ¹³ SSNMR spectrum as shown in FIG.

  In one embodiment, the invention relates to the tosylate salt of polymorph Form A compound (1). This form is the unsolvated form of the tosylate salt of compound (1). In one specific embodiment, the tosylate salt of Form A compound (1) is 7.2, 9.3, 13.7, 14.3, 14.7, 16. Characterized by one or more peaks corresponding to 2-theta values measured in units of 9, 18.7, 26.3 and 26.9 (± 0.2) degrees. In another specific embodiment, the tosylate salt of Form A compound (1) is measured in units of 6.0, 28.0 and 27.5 (± 0.2) degrees in a powder X-ray diffraction pattern. Further characterized by one or more peaks corresponding to the determined 2-theta values. In another specific embodiment, the tosylate salt of Form A compound (1) has an XRPD pattern with a characteristic peak represented by 2-theta ± 0.2 at the positions listed in Table 14 below: It is characterized by having In yet another specific embodiment, the tosylate salt of Form A compound (1) is characterized as having substantially the same XRPD pattern as shown in FIG. Their XRPD patterns are obtained at room temperature using CuK alpha radiation.

In another embodiment, the present invention provides a compound of Form A (1), HCl salt of 1 / 2H ₂ O, Form F of Compound (1), HCl of 3H ₂ O, Form D of Compound (1) The invention relates to a method for preparing the HCl salt, Form A compound (1) and the tosylate salt of Form A compound (1).

と定義され、式中、ｐは、その物質における水の蒸気圧であり、ｐ_０は、同じ温度の純水の蒸気圧であるか、またはａ_ｗ＝ｌ_ｗ×ｘ_ｗと定義され、式中、ｌ_ｗは、水の活量係数であり、ｘ_０は、水性画分における水のモル分率である。例えば、純水は、１．０という水分活性値を有する。水分活性値は、代表的には、静電容量式湿度計または露点式湿度計のいずれかによって得ることができる。様々なタイプの水分活性計測機器もまた商業的に入手可能である。あるいは、２つまたはそれを超える溶媒の混合物の水分活性値は、それらの溶媒の量およびそれらの溶媒の公知の水分活性値に基づいて算出され得る。 The HCl salt of Form A Compound (1) .1 / 2H ₂ O can be prepared by using a step of mixing (eg, stirring) hydrogen chloride (HCl) with Compound (1). Compound (1) may be solvated, unsolvated, amorphous, or crystalline. A solution, slurry or suspension of compound (1) may be mixed with HCl in a solvent system comprising water and one or more organic solvents, where the solvent system is equal to 0.05 or Beyond that and have a water activity equal to or less than 0.85, ie 0.05 to 0.85. The term “water activity” (a _w ) is used herein as is known in the art and means a measure of the energy state of water in a solvent system. It is defined as the vapor pressure of a liquid divided by the vapor pressure of pure water at the same temperature. Specifically, it is

Where p is the vapor pressure of water in the material and p ₀ is the vapor pressure of pure water at the same temperature or defined as a _w = l _w × x _w Where l _w is the activity coefficient of water and x ₀ is the molar fraction of water in the aqueous fraction. For example, pure water has a water activity value of 1.0. The water activity value can typically be obtained by either a capacitive hygrometer or a dew point hygrometer. Various types of water activity measuring instruments are also commercially available. Alternatively, the water activity value of a mixture of two or more solvents can be calculated based on the amount of those solvents and the known water activity value of those solvents.

  Examples of the crystalline compound (1) include A-form compound (1). Examples of solvates of compound (1) include 2-MeTHF, N, N-dimethylacetamide, N, N-dimethylformamide, methanol, xylene, acetone, 2-butanol, methyl acetate 1-pentanol, 2-propanol, tetrahydrofuran, methyltetrahydrofuran, dimethylacetamide N, N-dimethylformamide 1,4-dioxane, 1-pentanol, 2-methyl-1-propanol (2-methy-1-propanol) , Solvates of methyl ethyl ketone, 3-methyl-1-butanol, heptane, ethyl formate, 1-butanol, acetic acid and ethylene glycol. In a specific embodiment, a solvate of 2-MeTHF (eg, compound (1) · 1 (2-MeTHF)) is used.

Suitable solvent systems for the preparation of Form A compound (1) .1 / 2H ₂ O HCl salt may be composed of a wide variety of combinations of water and organic solvents, where the water activity of these solvent systems Is equal to or greater than 0.05 and less than or equal to 0.85 (0.05-0.85). In a specific embodiment, the water activity value is between 0.4 and 0.6. Suitable organic solvents include Class II or Class III organic solvents listed in the guidelines of the International Conference on Harmonization of Pharmaceutical Regulations. Specific examples of suitable class II organic solvents include chlorobenzene, cyclohexane, 1,2-dichloroethene, dichloromethane, 1,2-dimethoxyethane, N, N-dimethylacetamide, N, N-dimethylformamide, 1 , 4-dioxane, 2-ethoxyethanol, formamide, hexane, 2-methoxyethanol, methylbutylketone, methylcyclohexane, N-methylpyrrolidone, nitromethane, pyridine, sulfolane, tetrahydrofuran (THF), tetralin, toluene (toluene), 1 1,2-trichloroethene and xylene. Specific examples of suitable class III organic solvents include: acetic acid, acetone, anisole, 1-butanol, 2-butanol, butyl acetate, tert-butyl methyl ether, cumene, heptane, isobutyl acetate, isopropyl acetate, methyl acetate 3-methyl-1-butanol, methyl ethyl ketone, methyl isobutyl ketone, 2-methyl-1-propanol, ethyl acetate, ethyl ether, ethyl formate, pentane, 1-pentanol, 1-propanol, 2-propanol and propyl acetate Can be mentioned. In one specific embodiment, the organic solvent in the solvent system is chlorobenzene, cyclohexane, 1,2-dichloroethane, dichloromethane, 1,2-dimethoxyethane, hexane, 2-methoxyethanol, methylbutylketone, methylcyclohexane, Nitromethane, tetralin, xylene, toluene, 1,1,2-trichloroethane, acetone, anisole, 1-butanol, 2-butanol, butyl acetate, t-butyl methyl ether, cumene, ethanol, ethyl acetate, ethyl ether, ethyl formate, Heptane, isobutyl acetate, isopropyl acetate, methyl acetate, 3-methyl-1-butanol, methyl ethyl ketone, 2-methyl-1-propanol, pentane, 1-propanol, 1-pentanol, 2-propanol, propylene acetate It is selected from the group consisting of tetrahydrofuran and methyltetrahydrofuran. In another specific embodiment, the solvent-based organic solvent is 2-ethoxyethanol, ethylene glycol, methanol, 2-methoxyethanol, 1-butanol, 2-butanol, 3-methyl-1-butanol, 2- Methyl-1-propanol, ethanol, 1-pentanol, 1-propanol, 2-propanol, methyl butyl ketone, acetone, methyl ethyl ketone, methyl isobutyl ketone, butyl acetate, isobutyl acetate, isopropyl acetate, methyl acetate, ethyl acetate, propyl acetate , Pyridine, toluene and xylene. In yet another embodiment, the organic solvent is selected from the group consisting of acetone, n-propanol, isopropanol, isobutyl acetate and acetic acid. In yet another embodiment, the organic solvent is selected from the group consisting of acetone and isopropanol. In yet another specific embodiment, the solvent system comprises water and (an) acetone. In yet another specific embodiment, the solvent system comprises water and isopropanol.

Preparation of Form A Compound (1) .1 / 2H ₂ O HCl salt may be carried out at any suitable temperature. Typically, it is performed at a temperature of 5 ° C to 75 ° C. In a specific embodiment, it is performed at a temperature of 15 ° C to 75 ° C. In another specific embodiment, it is performed at a temperature between 15 ° C and 60 ° C. In yet another specific embodiment, it is performed at a temperature of 15C to 35C. In yet another specific embodiment, the preparation is performed at 5 ° C. to 75 ° C. in a solvent system having a water activity value of 0.4 to 0.6. In yet another specific embodiment, the preparation is performed at 15 ° C. to 75 ° C. in a solvent system having a water activity value of 0.4 to 0.6. In yet another specific embodiment, the preparation is performed at 15-60 ° C. in a solvent system having a water activity value of 0.4-0.6. In yet another specific embodiment, the preparation is performed at 15 ° C. to 35 ° C. in a solvent system having a water activity value of 0.4 to 0.6.

  Hydrogen chloride (HCl) can be charged as a solution or a gas. As one example, a suitable hydrogen chloride source is an aqueous solution of hydrogen chloride containing 30-40 wt% (eg, 34 wt% -38 wt%) HCl based on the weight of the aqueous solution.

The HCl salt of compound (1) .3H ₂ O in Form F can be prepared by mixing HCl and compound (1) in a solvent system containing water or containing water and one or more organic solvents. Where the solvent system has a water activity equal to or greater than 0.9 (≧ 0.9). The mixture can be a solution, slurry or suspension. Compound (1) may be solvated, unsolvated, amorphous, or crystalline. Alternatively, it can be prepared by stirring the HCl salt of Compound A (1) 1/2 H ₂ O in form A in a solvent system containing water or containing water and one or more organic solvents, wherein And the solvent system has a water activity equal to or greater than 0.9. Typically, pure water has a water activity value of 1.0. Thus, a solvent system having a water activity of 0.9-1.0 may be suitable for the preparation of the HCl salt of Form F compound (1) .3H ₂ O. In a specific embodiment, mixing or stirring is performed at ambient temperature (18 ° C. to 25 ° C.). In another specific embodiment, the mixing or stirring is performed at a temperature of 15 ° C to 30 ° C. In another specific embodiment, the mixing or stirring is performed at a temperature between 20 ° C. and 28 ° C. (eg, 25 ° C.). Suitable organic solvents (including specific examples) for forming the F salt of the F form of the compound (1) .3H ₂ O HCl include the specific form of the A form of the compound (1) .1 / 2H ₂ O HCl salt. As described above. In yet another specific embodiment, the solvent system comprises water and acetone. In yet another specific embodiment, the solvent system comprises water and isopropanol.

The HCl salt of Form D compound (1) can be prepared by dehydrating the HCl salt of Form A Compound (1) .1 / 2H ₂ O. The dehydration can be performed by any suitable means, such as a heating or dry nitrogen purge or both.

  Form A compound (1) can be obtained by mixing (a) a mixture of amorphous compound (1) or a solvate of compound (1) (for example, 2-MeTHF of compound (1) in a solvent system containing water and ethanol. Solvate) can be prepared by stirring. The mixture can be a solution or a slurry. In a specific embodiment, the stirring step is performed at a temperature in the range of 18 ° C to 90 ° C. In another specific embodiment, the stirring step (a) is performed at the reflux temperature of the solvent system. In another specific embodiment, the solvent system comprises 5 wt% to 15 wt% water based on the weight of the solvent system. Examples of solvates of compound (1) are as described above. In a specific embodiment, a solvate of 2-MeTHF (eg, compound (1) · 1 (2-MeTHF)) is used.

  In another embodiment, the method of preparing Form A compound (1) comprises (b) stirring amorphous form Compound (1) in nitromethane to form seed crystals of Form A Compound (1). And (c) further adding a seed crystal of the compound (1) in its A form to the mixture obtained in the mixing step (a). In a specific embodiment, the method comprises the steps of (b) stirring amorphous compound (1) in nitromethane to form seed crystals of Form A compound (1); (c) mixing step Cooling the mixture obtained in (a) to a temperature in the range of 18 ° C. to 60 ° C. (eg 50 to 55 ° C. or 55 ° C.); and (d) adding A to the mixture obtained in step (c). Adding a seed crystal of compound (1) in the form. In another specific embodiment, the method comprises, after addition of water, an amount of water such that the resulting solvent system will contain 15 to 25 wt% water, the seed of Form A compound (1) It further includes a step of adding to the mixture obtained through the refluxing step before adding the crystals. In yet another specific embodiment, the method comprises, after addition of water, an amount of water such that the resulting solvent system will contain 35 to 45 wt% water, compound A (1) Adding to the mixture containing the seed crystals. In yet another specific embodiment, the method further comprises cooling the mixture comprising seed crystals of Form A compound (1) after addition of water to a temperature between 0 ° C. and 10 ° C. .

  In one specific embodiment, seed crystals of Form A compound (1) may be prepared by 2-MeTHF solvate of compound (1) in nitromethane. In one embodiment, the solvent system for the reflux step comprises 5-15 wt% (eg, 8 wt%, 10 wt%, or 12 wt%) water based on the weight of the solvent system.

  Tosylate salt of Form A compound (1) can be obtained by using amorphous compound (1) or solvate of compound (1) (for example, 2-MeTHF solvate of compound (1)) and p-toluene. It can be prepared by stirring a mixture of a sulfonic acid and a solvent system comprising acetonitrile, hi a specific embodiment, the mixing or stirring step is performed at ambient temperature. The mixing or stirring step is performed at a temperature of 15 ° C. to 30 ° C. In another specific embodiment, the mixing or stirring step is performed at a temperature of 20 ° C. to 30 ° C. (eg, 25 ° C.). Suitable examples (including specific examples) of solvates of compound (1) are as described above for the preparation of Form A compound (1).

  In yet another embodiment, the invention relates to a 2-MeTHF solvate of compound (1). In one specific embodiment, the solvate is 0.5 to 1.5 equivalents of 2-MeTHF per compound (1), such as 1 equivalent of 2-MeTHF per compound (1). including. In one specific embodiment, the solvate contains 1 equivalent of 2-MeTHF and is in the following positions: 8.4, 9.7, 16.7, 16.9, 17.4, 21. Characterized as having an XRPD pattern with a characteristic peak represented by 2-theta ± 0.2 at 0, 22.3 and 25.7. In another specific embodiment, the solvate contains 1 equivalent of 2-MeTHF and has certain XRPD peaks listed in Table 12 or an XRPD pattern as shown in FIG. It is characterized by having.

  In yet another embodiment, the present invention provides amorphous compound (1) and pharmaceutically acceptable salts thereof (eg, amorphous HCl salt of compound (1) and amorphous compound (1 )). In yet another embodiment, the invention also encompasses Form B compound (1) hydrate. Form B compound (1) hydrate is isomorphic to Form A compound (1) and exhibits the same XRPD peak as the XRPD peak for Form A compound (1), but in the presence of water, for example, It is formed in a system having a water activity of greater than 0.6 (eg, 0.6 to 1.0) at ambient temperature.

  The present invention relates to Compound (I) or any other form of isolated form, pure form, or other material, such as other forms (ie, amorphous form, Form A compound (1), etc.) And the polymorphic compound (1) described above in a mixture as a solid composition when admixed with the above materials.

In one embodiment, the present invention provides polymorphs, for example, Form A compound (1), HCl salt of 1 / 2H ₂ O, Form F compound (1), HCl salt of 3H ₂ O, Form D Provided are the HCl salt of (1), Form A compound (1), Form B compound (1) hydrate and Form A compound (1) tosylate salt in the form of an isolated solid. In yet another aspect, the present invention provides an amorphous form of compound (1) and pharmaceutically acceptable salts thereof, such as amorphous HCl salt of compound (1) and amorphous compound (1). Is provided in the form of an isolated solid.

In a further aspect, the present invention provides polymorphs, for example, Form A compound (1), HCl salt of 1 / 2H ₂ O, Form F compound (1), HCl salt of 3H ₂ O, Form D compound ( The HCl salt of 1), Form A compound (1), Form B compound (1) hydrate and Form A compound (1) tosylate salt are provided in pure form. Its pure form is that the particular polymorph is more than 95% (w / w), for example, more than 98% (w / w), more than 99% (w / w%), 99.5% (w / w) ) Or more than 99.9% (w / w). In another further aspect, amorphous form of Compound (1) or a pharmaceutically acceptable salt thereof is provided in pure form. The pure form has an amorphous form of greater than 95% (w / w), eg, greater than 98% (w / w), greater than 99% (w / w%), 99.5% (w / w) Mean greater than or greater than 99.9% (w / w).

More specifically, the invention relates to polymorphs in the form of polymorphic compositions or mixtures comprising one or more other crystalline forms, solvates, amorphous or other polymorphs, or combinations thereof. Provide each of. For example, in one embodiment, the composition comprises an HCl salt of Form A Compound (1) .1 / 2H ₂ O with one or more other polymorphs of Compound (1) (eg, amorphous form) , Solvates, HCl salts of Form D compound (1), Form F compound (1), HCl salt of 3H ₂ O, Form A compound (1) and / or other forms or any combination thereof ) Similarly, in another embodiment, the composition comprises an HCl salt of Form F Compound (1) .3H ₂ O with one or more other polymorphs of Compound (1) (eg, amorphous form, Solvates, Form A Compound (1), HCl salt of 1 / 2H ₂ O, Form D Compound (1) HCl salt, Form A Compound (1) and / or other forms or combinations thereof) Include with. Similarly, in another embodiment, the composition comprises the HCl salt of Form D compound (1) with one or more other polymorphs of compound (1) (eg, amorphous form, solvate, Compound A in Form A (1). HCl salt of 1 / 2H ₂ O, Compound F in Form F (1), HCl salt of 3H ₂ O, Compound A in Form A (1) and / or other forms or combinations thereof Include with. In yet another embodiment, the composition comprises Compound A (1) in Form A and one or more other polymorphs of Compound (1) (eg, amorphous forms, hydrates, solvates and / Or other forms or combinations thereof). In yet another embodiment, the composition comprises a tosylate salt of Form A Compound (1) with one or more other polymorphs of Compound (1) (eg, amorphous form, hydrate, solvent Japanese and / or other forms or combinations thereof). More specifically, the composition is based on the total amount of compound (1) in the composition, from trace amounts to 100%, or any amount, such as 0.1% to 0.5% by weight. 0.1 wt% to 1 wt%, 0.1 wt% to 2 wt%, 0.1 wt% to 5 wt%, 0.1 wt% to 10 wt%, 0.1 wt% to 20 wt% Specific polymorphs in the range of 0.1 wt% to 30 wt%, 0.1 wt% to 40 wt%, or 0.1 wt% to 50 wt%. Alternatively, the composition is at least 50%, 60%, 70%, 80%, 90%, 95%, 97% by weight based on the total amount of compound (1) in the composition , 98%, 99%, 99.5% or 99.9% by weight of a particular polymorph.

  For the purposes of the present invention, chemical elements are described in Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Specified according to. Furthermore, the general principles of organic chemistry are described in “Organic Chemistry”, Thomas Sorell, University Science Books, Sausolito: 1999 and “March's Advanced Organic Chemistry”, 5th. Ed. Smith, M .; B. and March, J.A. , John Wiley & Sons, New York: 2001, the entire contents of which are incorporated herein by reference.

として描かれる置換基は、

にも相当する。 Unless otherwise indicated, structures depicted herein include all isomers of the structure (eg, enantiomers, diastereoisomers, cis-trans isomers, conformers and rotamers). It is also meant to include. For example, only one of the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformers is clearly depicted. As long as these isomers are included in the present invention. As can be appreciated by those skilled in the art, the substituents are free to rotate around any rotatable bond. For example,

The substituents depicted as

It corresponds to.

  Thus, single stereochemical isomers of the present compounds, as well as enantiomeric, diastereomeric, cis / trans isomer, conformational and rotamer mixtures of the present invention Within range.

  Unless otherwise indicated, all tautomeric forms of the compounds of the invention are within the scope of the invention.

Further, unless otherwise indicated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having this structure other than replacement of hydrogen with deuterium or tritium, or replacement of carbon with carbon enriched in ¹³ C or ¹⁴ C are within the scope of the present invention. Such compounds are useful, for example, as probes in analytical tools or biological assays. Such compounds, particularly deuterium analogs, can also be useful therapeutically.

  The compounds described herein are defined by their chemical structure and / or chemical name. If a compound is referred to by both chemical structure and chemical name and the chemical structure and chemical name conflict, the chemical structure determines what the compound is.

  It will be appreciated by those skilled in the art that the compounds according to the invention may contain chiral centers. Thus, the compounds of those formulas may exist in the form of two different optical isomers (ie, (+) or (−) enantiomers). All such enantiomers and mixtures thereof including racemic mixtures are included within the scope of the present invention. Single optical isomers or enantiomers can be obtained by methods well known in the art (eg, chiral HPLC, enzymatic resolution and chiral auxiliaries).

  In one embodiment, the compounds according to the invention do not contain the corresponding enantiomer and are provided in the form of at least 95%, at least 97% and at least 99% single enantiomer.

  In a further embodiment, the compounds according to the invention do not contain the corresponding (−) enantiomer and exist in the form of at least 95% of the (+) enantiomer.

  In a further embodiment, the compounds according to the invention do not contain the corresponding (−) enantiomer and exist in the form of at least 97% of the (+) enantiomer.

  In a further embodiment, the compounds according to the invention do not contain the corresponding (−) enantiomer and exist in the form of at least 99% of the (+) enantiomer.

  In a further embodiment, the compounds according to the invention do not comprise the corresponding (+) enantiomer and exist in the form of at least 95% (−) enantiomer.

  In a further embodiment, the compounds according to the invention do not contain the corresponding (+) enantiomer and exist in the form of at least 97% (−) enantiomer.

  In a further embodiment, the compounds according to the invention do not contain the corresponding (+) enantiomer and exist in the form of at least 99% (−) enantiomer.

  II. Use of compound (1) and pharmaceutically acceptable salts thereof

One aspect of the present invention is generally to inhibit influenza virus replication in a biological sample or patient, to reduce the amount of influenza virus in the biological sample or patient (to reduce viral titer), And various solid forms described above (eg, HCl salt of Form A (1) .1 / 2H ₂ O, Form F (1) .3H ₂ ) to treat influenza in patients and patients. HCl salt of O, HCl salt of D-form compound (1), A-form compound (1), B-form compound (1) hydrate and A-form compound (1) tosylate salt) 1) and the use of pharmaceutically acceptable salts thereof. Herein after, unless otherwise indicated specifically, the form of various solids listed above (e.g., A form of Compound (1) · 1 / 2H 2 O of HCl salt, F form of Compound (1 ) · HCl salt of 3H ₂ O, HCl salt of D-form compound (1), A-form compound (1), B-form compound (1) hydrate and A-form compound (1) tosylate salt) Compound (1) and pharmaceutically acceptable salts thereof, are broadly referred to for compounds.

  In one embodiment, the present invention generally relates to a compound disclosed herein (eg, a compound in a pharmaceutically acceptable composition) for any of the uses specified above. About the use of.

  In yet another embodiment, a compound disclosed herein is a viral titer in a biological sample (eg, an infected cell culture) or a human titer (eg, a lung viral titer in a patient). ).

  The terms “influenza virus mediated condition”, “influenza infection” or “influenza” as used herein are used interchangeably to mean a disease caused by infection with an influenza virus. The

  Influenza is an infection that occurs in birds and mammals caused by influenza viruses. Influenza viruses are RNA viruses of the Orthomyxoviridae family that includes five genera: influenza virus A, influenza virus B, influenza virus C, ISA virus and Togotovirus. Influenza virus A has one species, influenza A virus, which can be subdivided into different serotypes based on antibody responses to these viruses: H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3 and H10N7. Further examples of influenza A virus include H3N8 and H7N9. Influenza virus B has one species, the influenza B virus. Influenza B almost exclusively infects humans and is less common than influenza A. The influenza virus C genus has one species, influenza C virus, which can infect humans and pigs, causing severe illness and regional epidemics. However, influenza virus C is less common than the other types and usually appears to cause mild disease in children.

  In some embodiments of the invention, the influenza or influenza virus is associated with influenza virus A or B. In some embodiments of the invention, the influenza or influenza virus is associated with influenza virus A. In some specific embodiments of the invention, influenza virus A is H1N1, H2N2, H3N2, or H5N1. In some specific embodiments of the invention, influenza virus A is H1N1, H3N2, H3N8, H5N1 and H7N9. In some specific embodiments of the invention, influenza virus A is H1N1, H3N2, H3N8 and H5N1.

  In humans, common symptoms of influenza are chills, fever, sore throat, myalgia, severe headache, cough, weakness and general discomfort. In more severe cases, influenza causes pneumonia, which can be fatal, especially in younger children and the elderly. Influenza is often confused with the common cold, but is even more severe and is caused by different types of viruses. Influenza can cause nausea and vomiting, particularly in children, but these symptoms are characteristic of unrelated gastroenteritis, which is sometimes referred to as “viral stomach flu” or “24-hour flu” (24-hour flu) ".

  Influenza symptoms can begin fairly suddenly 1-2 days after infection. Usually the first symptom is chills or chills, but fever is also common early in the infection, with body temperatures ranging from 38 ° C to 39 ° C (approximately 100 ° F to 103 ° F). Many people are sick enough to lie down on a bed for several days, with pain and pain throughout the body (more bad in the back and legs). Symptoms of influenza include body pain, especially joint and throat pain, extreme coldness and fever, fatigue, headache, irritation and tearing eyes, red eyes, skin (especially the face) , Mouth, pharynx and nose, abdominal pain (in children with influenza B). Influenza symptoms are non-specific and overlap with many pathogens ("influenza-like illness"). Usually, test data is needed to confirm the diagnosis.

  The terms “disease”, “disorder” and “symptom” may be used interchangeably herein to refer to a medical or pathological symptom mediated by influenza virus.

  As used herein, the terms “subject” and “patient” are used interchangeably. The terms “subject” and “patient” refer to animals (eg, birds, such as chickens, quails or turkeys, or mammals), specifically non-primates (eg, cows, pigs, horses, sheep, “Mammals” including rabbits, guinea pigs, rats, cats, dogs and mice) and primates (eg monkeys, chimpanzees and humans), more specifically humans. In one embodiment, the subject is a non-human animal, such as a domestic animal (eg, horse, cow, pig or sheep) or pet (eg, dog, cat, guinea pig or rabbit). In preferred embodiments, the subject is a “human”.

  The term “biological sample” as used herein refers to a cell culture or extract thereof; a biopsy material obtained from a mammal or an extract thereof; blood, saliva, urine, stool, semen , Including but not limited to tears or other body fluids or extracts thereof.

  As used herein, “infection efficiency” or “MOI” is the ratio of an infectious agent (eg, a phage or virus) to an infection target (eg, a cell). For example, when referring to a group of cells inoculated with infectious virus particles, the infection efficiency or MOI is calculated by the number of infectious virus particles deposited in the well as the number of target cells present in that well. A ratio defined by dividing.

  As used herein, the term “inhibition of influenza virus replication” refers to a reduction in the amount of viral replication (eg, a reduction of at least 10%) and a complete cessation of viral replication (ie, an amount of viral replication). 100% reduction). In some embodiments, influenza virus replication is inhibited by at least 50%, at least 65%, at least 75%, at least 85%, at least 90% or at least 95%.

  Influenza virus replication can be measured by any suitable method known in the art. For example, an influenza virus titer in a biological sample (eg, an infected cell culture) or an influenza virus titer in a human (eg, a lung virus titer in a patient) can be measured. More specifically, in the case of cell-based assays, in each case, the cells are cultured in vitro, the virus is added to the culture in the presence or absence of the test substance, and a suitable length of time. After the elapse, virus-dependent endpoints are evaluated. For a typical assay, Madin-Darby canine kidney cells (MDCK) and A / Puerto Rico / 8/34, an influenza strain adapted to standard tissue culture, can be used. A first type of cellular assay that can be used in the present invention is a process called cytopathic effect (CPE), where viral infection causes depletion of cell resources and eventual lysis of the cells. Depends on the killing of infected target cells. In this first type of cell assay, a low percentage of cells in the wells of a microtiter plate are infected (usually 1/10 to 1/1000) and the virus is replicated several times over 48 to 72 hours, The amount of cell death is then measured using the decrease in the ATP content of the cell as compared to the non-infected control. A second type of cellular assay that can be used in the present invention relies on mitotic growth of virus-specific RNA molecules in infected cells, where RNA levels use a branched DNA hybridization method (bDNA). Measured directly. In this second type of cell assay, a small number of cells in the wells of a microtiter plate are first infected, the virus is replicated in the infected cells and spread to further generations of cells, and Cells are lysed and the viral RNA content is measured. This assay is stopped early, usually after 18-36 hours, while all target cells are viable. Viral RNA is quantified by amplification of the signal by hybridization to a specific oligonucleotide probe immobilized in the well of the assay plate, followed by hybridization with an additional probe linked to a reporter enzyme.

As used herein, “virus titer (or titer)” is a measure of virus concentration. A titer test can obtain approximate quantitative information from an analytical procedure that essentially evaluates only positive or negative by using serial dilutions. This titer still corresponds to the highest dilution factor that yields a positive reading; for example, a positive reading in the first 8 of a 2-fold serial dilution translates to a titer of 1: 256. A specific example is the virus titer. In order to measure its titer, several dilutions (eg 10 ⁻¹ , 10 ⁻² , 10 ⁻³ , 10 ⁻⁴ , 10 ⁻⁵ , 10 ⁻⁶ , 10 ⁻⁷ , 10 ⁻⁸ ) Prepared. Still, the lowest virus concentration that infects cells is the virus titer.

  As used herein, the terms “treat”, “treatment” and “treating” refer to both therapeutic and prophylactic treatment. For example, therapeutic treatments include progression or reduction in severity and / or duration of symptoms mediated by influenza virus, or one or more therapies (eg, one or more therapeutic agents Of one or more of the symptoms mediated by influenza virus (specifically, one or more identifiable symptoms) resulting from the administration of, for example, a compound or composition of the invention) Includes recovery. In certain embodiments, the therapeutic treatment includes restoration of at least one measurable physical parameter of symptoms mediated by influenza virus. In other embodiments, the therapeutic treatment includes influenza virus, either physically (eg, by stabilizing identifiable symptoms), physiological (eg, by stabilizing physical parameters), or both. Inhibition of the progression of symptoms mediated by. In other embodiments, the therapeutic treatment includes the reduction or stabilization of an infection mediated by influenza virus. Antiviral drugs can be used in the social environment to treat people who already have influenza to reduce the severity of symptoms and to reduce the number of unwell days.

  The term “chemotherapy” refers to the use of an agent, such as a small molecule drug (not a “vaccine”), to treat a disorder or disease.

  The terms “prophylaxis” or “prophylactic use” and “prophylactic treatment” as used herein are any medicine whose purpose is to prevent rather than treat or cure a disease. Refers to traditional or public health procedures. As used herein, the terms “prevent”, “prevention” and “preventing” are not currently a disease but are close to a person who had the disease. Refers to a reduction in the risk of acquiring or developing a given symptom, or recurrence or a reduction or inhibition of said symptom in a subject who has been or may have been near. The term “chemoprophylaxis” refers to the use of a drug, such as a small molecule drug (not a “vaccine”), to prevent a disorder or disease.

  As used herein, prophylactic use includes a place where a large number of people at high risk of severe influenza complications live in close contact with each other (eg, wards, day care centers, prisons, Use in situations where a pandemic is detected to prevent contact infection or spread of infection in a nursing home, etc.). Prophylactic use requires protection from influenza but is not protected after vaccination (eg due to weak immune system) between populations, or the population Also included is use between populations when they are not available or when the population cannot receive a vaccine due to side effects. Since the two weeks after vaccination is the time that the vaccine is still ineffective, prophylactic use includes use during that two weeks. Prophylactic use reduces the probability that a person who does not have flu or is not considered at high risk of complications will be infected with the flu, and the person (eg, health care worker, nursing home worker, etc.) Treatment of people who do not have the flu or are not considered at high risk of complications may be included to reduce the probability of transmission to high-risk individuals who are in close contact.

  According to the US CDC, influenza “outbreaks” are a percentage of normal background in groups of people close to each other (eg, people in the same area of a nursing home, people in the same household, etc.) Defined as a sudden increase in acute febrile respiratory disease (AFRI) occurring within 48-72 hours when any subject in the population analyzed above or in the analyzed population tests positive for influenza. One case of influenza confirmed by any testing method is considered a pandemic.

  A “cluster” is an AFRI of 3 or more cases that occur within 48-72 hours in a group of people in close proximity (eg, people in the same area of a nursing home, people in the same household, etc.) Defined as a group of

  As used herein, the “index case”, “first case” or “patient number 1” is the first patient in the epidemiological population sample. When widely used to refer to such patients in epidemiological studies, the term is not capitalized. When the term is used to refer to a specific person instead of the person's name in a report about a specific study, the term is capitalized as Patient Zero . Often, scientists seek index cases to find out how the disease spreads and which reservoirs hold the disease during an outbreak. Note that the index case is the first patient showing the appearance of a pandemic. Earlier cases may be found and they are named first, second, third, etc.

  In one embodiment, the methods of the invention are prophylactic or “preemptive” measures for patients, particularly humans, predisposed to complications resulting from infection with influenza virus. For example, the terms “preemptive” or “preemptive”, as in “preemptive” use, as used herein, a community in a situation where “index cases” or “outbreaks” have been identified. Or prophylactic use to prevent the spread of infection to the rest of the population.

  In another embodiment, the methods of the invention are used as a “preemptive” measure to members of a community or population group, specifically humans, to prevent the spread of infection.

  As used herein, “effective amount” refers to an amount sufficient to elicit the desired biological response. In the present invention, the desired biological response is to inhibit influenza virus replication, to reduce the amount of influenza virus, or to reduce or recover the severity, duration, progression or occurrence of influenza virus infection. Prevent, treat, prevent the progression of influenza virus infection, prevent the recurrence, onset, development or progression of symptoms associated with influenza virus infection, or another treatment used for influenza infection To enhance or improve the effect (s). The exact amount of compound administered to a subject will depend on the mode of administration, the type and severity of the infection, and the characteristics of the subject (eg, general health, age, sex, weight and tolerance to drugs). . Persons of ordinary skill in the art can determine appropriate dosages depending on these and other factors. When co-administered with another antiviral agent, eg, when co-administered with an anti-influenza agent, the “effective amount” of the second agent depends on the type of drug used. Suitable dosages are known for approved drugs and depend on the condition of the subject, the type of symptom (s) being treated and the amount of compound described herein being used. Can be adjusted by those skilled in the art. If the amount is not explicitly stated, an effective amount should be assumed. For example, the compounds disclosed herein can be administered to a subject within a dosage range of approximately 0.01-100 mg / kg body weight / day for therapeutic or prophylactic treatment. .

  In general, the dosage regimen is the disorder being treated and the severity of the disorder; the activity of the specific compound being used; the specific composition being used; the age, weight, general health status of the patient. , Sex and diet; administration time, route of administration and excretion rate of the particular compound used; function of the kidney and liver of the subject; and the particular compound or salt thereof being used; duration of treatment; The drug may be selected according to a variety of factors including drugs used in combination with or simultaneously with the particular compound being considered, as well as similar factors well known in the medical field. Those skilled in the art will recognize that an effective amount of a compound described herein is necessary to treat, prevent, inhibit (completely or partially) the disease, or to stop the progression of the disease. Can be easily determined and prescribed.

  The dosage of the compounds described herein is 0.01-100 mg / kg body weight / day, 0.01-50 mg / kg body weight / day, 0.1-50 mg / kg body weight / day or 1-25 mg. / Kg body weight / day range. The total daily dose can be administered in a single dose, or multiple doses (eg, twice a day (eg, every 12 hours), three times a day (eg, every 8 hours), or 4 times a day It is understood that it can be administered once (eg every 6 hours).

In some embodiments, the compounds described herein (eg, compound (1) and pharmaceutically acceptable salts thereof (eg, various solid forms (eg, compound A (1) / 2H ₂ O HCl salt, Form F compound (1), HCl salt of 3H ₂ O, Form D compound (1) HCl salt, Form A compound (1), Form B compound (1) water The dose of the compound (including tosylate salt of Japanese and Form A compound (1)) is in the range of 100 mg to 1,600 mg, such as 400 mg to 1,600 mg or 400 mg to 1,200 mg. Can be taken once a day (QD), twice a day (eg every 12 hours (BID)) or three times a day (eg q8h (TID)) Any of QD, BID and TID The desired combination Also note that, for example, QD can be used after the first day BID.

In some embodiments, the compounds described herein (eg, compound (1) and pharmaceutically acceptable salts thereof (eg, various solid forms (eg, compound A (1) / 2H ₂ O HCl salt, F form compound (1), 3H ₂ O HCl salt, and D form compound (1) HCl salt) doses of 100 mg to 1,600 mg, for example, Within the range of 400 mg to 1,600 mg or 400 mg to 1200 mg, each dose is once a day (QD), twice a day (eg every 12 hours (BID)) or three times a day (Eg, q8h (TID)) Any combination of QD, BID and TID can be administered as desired, eg, BID on day 1 followed by QD, or on day 1 Amount is used Note that the QD can be used after the BID on the second day.

  In one specific embodiment, the dosage of the compounds described herein is 400 mg to 1,600 mg, 400 mg to 1,200 mg or 600 mg to 1,200 mg once daily. In another specific embodiment, the dosage of the compounds described herein is 400 mg to 1,600 mg, 400 mg to 1,200 mg or 300 mg to 900 mg twice a day. In yet another specific embodiment, the dosage of the compounds described herein is 400 mg to 1,000 mg once daily. In yet another specific embodiment, the dosage of the compounds described herein is from 600 mg to 1,000 mg once daily. In yet another specific embodiment, the dosage of the compounds described herein is from 600 mg to 800 mg once daily. In yet another specific embodiment, the dosage of the compounds described herein is 400 mg to 800 mg twice a day (eg, 400 mg to 800 mg every 12 hours). In yet another specific embodiment, the dosage of the compounds described herein is from 400 mg to 600 mg twice a day.

  In some embodiments, a loading dosing regimen is used. In one specific embodiment, a loading of 400 mg to 1,600 mg is used on the first day of treatment. In another specific embodiment, a loading of 600 mg to 1,600 mg is used on the first day of treatment. In another specific embodiment, a loading of 800 mg to 1,600 mg is used on the first day of treatment. In yet another specific embodiment, a loading of 900 mg to 1,600 mg is used on the first day of treatment. In yet another specific embodiment, a loading of 900 mg to 1,200 mg is used on the first day of treatment. In yet another specific embodiment, a 900 mg loading is used on the first day of treatment. In yet another specific embodiment, a loading of 1,000 mg is used on the first day of treatment. In yet another specific embodiment, a loading dose of 1,200 mg is used on the first day of treatment.

  In one specific embodiment, the dosage regimen of the compounds described herein is a loading dose of 600 mg to 1,600 mg on day 1 and 300 mg to 1, over the remainder of the treatment period. A fixed dose of 200 mg is used. Each defined dose may be taken once daily, twice daily, three times daily, or any combination thereof. In more specific embodiments, a loading dose of 900 mg to 1,600 mg (eg, 900 mg, 1,200 mg or 1,600 mg) is used. In another more specific embodiment, a loading dose of 900 mg to 1,200 mg (eg, 900 mg or 1,200 mg) is used. In yet another more specific embodiment, a defined dose of 400 mg to 1200 mg (400 mg, 600 mg or 800 mg) is used for the remainder of the treatment period. In yet another more specific embodiment, a defined dose of 400 mg to 1,000 mg over the remainder of the treatment. In yet another more specific embodiment, a defined dose of 400 mg to 800 mg is used for the remainder of the treatment period. In yet another more specific embodiment, a fixed dose of 300 mg to 900 mg twice a day is used. In yet another more specific embodiment, a fixed dose of 600 mg to 1,200 mg once a day is used. In yet another more specific embodiment, a fixed dose of 600 mg twice daily on the second day, then 600 mg once daily for the remainder of the treatment period.

  For therapeutic treatment, the compounds described herein are, for example, within 48 hours of the occurrence of symptoms (eg, nasal congestion, sore throat, cough, pain, fatigue, headache and chills / sweat) ( Or within 40 hours, or less than 2 days, or less than 1.5 days, or within 24 hours). Alternatively, for therapeutic treatment, the compounds described herein can be administered to a patient, for example, within 96 hours of the onset of symptoms. The therapeutic treatment can be continued for any suitable period of time, eg, 3 days, 4 days, 5 days, 7 days, 10 days, 14 days, etc. For prophylactic treatment during community outbreaks, the compounds described herein can be administered to patients in index cases, for example, within 2 days of symptom onset, any suitable Over a period of time, such as 7 days, 10 days, 14 days, 20 days, 28 days, 35 days, 42 days, etc., can be continued until the entire influenza season. The flu season is a recurrent year that is characterized by the spread of the influenza pandemic. Influenza activity can sometimes be predicted and even geographically tracked. The onset of major flu activity in each season varies from place to place, but in any particular place, these small epidemics usually take 3-4 weeks to peak and are significantly reduced It takes another 3-4 weeks to complete. Typically, the Centers for Disease Control (CDC) collects, aggregates and analyzes information on influenza activity throughout the year in the United States and produces a weekly report from October to mid-May.

  In one embodiment, the therapeutic treatment continues from one day to the entire flu season. In one specific embodiment, the therapeutic treatment lasts from 3 days to 14 days. In another specific embodiment, the therapeutic treatment lasts from 5 days to 14 days. In another specific embodiment, the therapeutic treatment lasts from 3 days to 10 days. In yet another specific embodiment, the therapeutic treatment continues for 4-10 days. In yet another specific embodiment, the therapeutic treatment continues for 5-10 days. In yet another specific embodiment, the therapeutic treatment continues for 4 days to 7 days (eg, 4 days, 5 days, 6 days or 7 days). In yet another specific embodiment, the therapeutic treatment continues for 5 to 7 days (eg, 5 days, 6 days or 7 days). In one specific embodiment, prophylactic treatment continues until the entire influenza season.

  In one specific embodiment, the compounds described herein have a loading dose of 900 mg to 1,600 mg on day 1 over 3 days to 14 days (eg, 5 days to 14 days), And is administered to the patient at a defined dose of 300 mg to 1,200 mg over the remainder of the treatment period. In another specific embodiment, the compound described herein has a loading dose of 900 mg to 1,200 mg on day 1 over 3 days to 14 days (eg, 5 days to 14 days), And is administered to the patient at a defined dose of 400 mg to 1,000 mg over the remainder of the treatment period. In yet another specific embodiment, the compound described herein is administered at a dose of 900 mg to 1,200 mg on day 1 for 3 days to 14 days (eg, 5 days to 14 days). The dose is administered to the patient at a fixed dose of 400 mg to 800 mg over the remainder of the treatment period. In yet another specific embodiment, the compound described herein is administered at a dose of 900 mg to 1,200 mg on day 1 for 3 days to 14 days (eg, 5 days to 14 days). The dose is administered to the patient at a fixed dose of 400 mg to 800 mg over the remainder of the treatment period. Each dose may be taken once daily, twice daily or three times daily, or any combination thereof.

  In one specific embodiment, the compound described herein has a loading dose of 900 mg to 1,600 mg on day 1 over 3 days to 14 days, and over the remainder of the treatment period. It is administered to patients at a fixed dose of 600 mg to 1,000 mg once a day. In another specific embodiment, the compound described herein has a loading dose of 900 mg to 1,200 mg on day 1 over 3 days to 14 days, and over the remainder of the treatment period. The patient is administered a fixed dose of 600 mg to 800 mg once daily (eg, 600 mg, 650 mg, 700 mg, 750 mg or 800 mg). In some embodiments, the treatment period is from 4 days to 10 days, from 5 days to 10 days, or from 5 days to 7 days.

  In one specific embodiment, the compound described herein has a loading dose of 900 mg to 1,600 mg on day 1 over 3 days to 14 days, and over the remainder of the treatment period. It is administered to patients at a fixed dose of 400 mg to 800 mg twice a day. In another specific embodiment, the compound described herein has a loading dose of 900 mg to 1,200 mg on day 1 over 3 days to 14 days, and over the remainder of the treatment period. The patient is administered a fixed dose of 400 mg to 600 mg (e.g., 400 mg, 450 mg, 500 mg, 550 mg or 600 mg) twice daily. In some embodiments, the period is 4 days to 10 days, 5 days to 10 days, or 5 days to 7 days.

  In one specific embodiment, a compound described herein has a loading dose of 900 mg to 1,200 mg (eg, 900 mg or 1,200 mg) on day 1 for 4 days or 5 days, And is administered to the patient at a defined dose of 400 mg to 600 mg (eg, 400 mg or 600 mg) twice a day for the remainder of the treatment period (eg, days 2-4 or 2-5). In another specific embodiment, a compound described herein has a loading dose of 900 mg to 1,200 mg (eg, 900 mg or 1,200 mg) on day 1 for 4 days or 5 days, And is administered to the patient at a fixed dose of 600 mg to 800 mg (e.g., 600 mg or 800 mg) once a day for the remainder of the treatment period.

  Various types of administration methods can be used in the present invention and are described in detail in the section entitled “Methods of Administration” below.

  Various types of administration methods can be used in the present invention and are described in detail in the section entitled “Methods of Administration” below.

  III. Combination therapy

  An effective amount is a compound of the invention (including pharmaceutically acceptable salts or solvates (eg, hydrates)) alone or in combination with a suitable additional therapeutic agent, eg, an antiviral agent or vaccine. In the method or pharmaceutical composition of the invention used. When “combination therapy” is used, an effective amount can be achieved using a first amount of a compound of the invention and a second amount of a suitable additional therapeutic agent (eg, an antiviral agent or vaccine).

  In another embodiment of the invention, the compound of the invention and the additional therapeutic agent are each administered in an effective amount (ie, an amount that is therapeutically effective when each is administered alone). In another embodiment, the compound of the invention and the additional therapeutic agent are each administered in an amount that does not produce a therapeutic effect alone (sub-therapeutic dose). In yet another embodiment, the compounds of the invention can be administered in an effective amount and the additional therapeutic agent is administered at a sub-therapeutic dose. In yet another embodiment, the compounds of the invention can be administered at sub-therapeutic doses, with additional therapeutic agents, eg, suitable cancer therapeutic agents, administered in an effective amount.

  As used herein, the term “in combination” or “co-administration” refers to the use of more than one treatment (eg, one or more prophylactic and / or therapeutic agents). Can be used interchangeably to refer. The use of these terms does not limit the order in which treatments (eg, prophylactic and / or therapeutic agents) are administered to a subject.

  Co-administration is in essentially the same manner (eg, a single pharmaceutical composition having a fixed ratio of the first amount and the second amount, eg, a capsule or tablet) or to each Including co-administration of a first amount and a second amount of a compound in a plurality of separate capsules or tablets. Furthermore, such co-administration also encompasses the use of each compound in a sequential manner in any order.

  In one embodiment, the present invention uses the compounds described herein to inhibit influenza virus replication in a biological sample or patient or to treat influenza virus infection in a patient. It relates to a method of combination therapy for prevention or prevention. Accordingly, the pharmaceutical composition of the present invention also includes a composition comprising the influenza virus replication inhibitor of the present invention together with an antiviral compound exhibiting anti-influenza virus activity.

  Methods of using the compounds described herein and the compositions of the invention include a combination of chemotherapy with a compound or composition of the invention, or another antiviral agent with the compound or composition of the invention and Including combined use with vaccination with influenza vaccine.

  When co-administration includes separate administration of a first amount of a compound of the invention and a second amount of a further therapeutic agent, the compounds are administered at a time sufficiently close to provide the desired therapeutic effect. The For example, the length of time between each administration that can produce the desired therapeutic effect can range from minutes to hours, with the properties of each compound (eg, potency, solubility, bioavailability, plasma half-life and kinetics) A dynamic profile). For example, the compound of the invention and the second therapeutic agent can be within 24 hours of each other, within 16 hours of each other, within 8 hours of each other, within 4 hours of each other, within 1 hour of each other, or They can be administered in any order within 30 minutes of each other.

  More specifically, a first treatment (eg, a prophylactic or therapeutic agent, eg, a compound of the present invention) administers a second treatment (eg, a prophylactic or therapeutic agent, eg, an anticancer agent) to a subject. Before (for example, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 Time, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks), at the same time or after the administration (For example, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours later 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks or 12 weeks) It may be given.

  It will be appreciated that the method of co-administration of a first amount of a compound of the invention and a second amount of a further therapeutic agent can provide an enhanced therapeutic effect or a synergistic therapeutic effect, wherein the combination The combined effect is greater than the additive effect resulting from the separate administration of a first amount of a compound of the invention and a second amount of a further therapeutic agent.

  As used herein, the term “synergistic” refers to a combination of a compound of the invention and another treatment (eg, a prophylactic or therapeutic agent), which It is more effective than the additive effect. Synergistic effects of treatment combinations (eg, prophylactic or therapeutic agent combinations) can be achieved by using one or more of those treatments at lower doses, and / or less frequently such treatment to the subject It may be possible to administer. The use of lower dose therapies (eg, prophylactic or therapeutic agents) and / or the less frequent administration of the therapies can increase the effectiveness of the treatment in preventing, managing or treating disorders. Without reducing, the toxicity associated with administering the treatment to a subject can be reduced. Furthermore, synergistic effects can result in improved efficacy of the drug in preventing, managing or treating the disorder. Ultimately, the synergistic effect of a therapeutic combination (eg, a prophylactic or therapeutic combination) may avoid the harmful or unwanted side effects associated with using either treatment alone, Or it can be reduced.

  When a combination therapy using a compound of the invention is used in combination with an influenza vaccine, the time between each administration can be longer (eg, days, weeks or months) A therapeutic agent can be administered.

  The presence of a synergistic effect can be determined using a method suitable for assessing drug interactions. Suitable methods include, for example, the Sigmaid-Emax equation (Holford, NHG and Scheiner, LB, Clin. Pharmacokinet. 6: 429-453 (1981)), the Loewe addition equation (Loewe, S And Muischnek, H., Arch.Exp.Pathol Pharmacol.114: 313-326 (1926)) and the median-effect equation (Chou, TC and Talalay, P., Adv. Enzyme Regul). 22: 27-55 (1984)). Each equation mentioned above can be used for experimental data to create a corresponding graph, which is useful for evaluating the effects of drug combinations. The corresponding graphs associated with the equations referred to above are the concentration-effect curve, isobologram curve and combination index curve, respectively.

  Specific examples that may be co-administered with the compounds described herein include neuraminidase inhibitors (eg, oseltamivir (Tamiflu®) and zanamivir (Rlenza®)), viral ion channels ( M2 protein) blockers (e.g. amantadine (Symmetrel (R)) and rimantadine (Flumadine (R))) and described in WO2003 / 015798 including T-705 under development by Toyama Chemical in Japan Antiviral drugs (Ruruta et al., Antibiotic Research, 82: 95-102 (2009), “T-705 (flavipiravir) and related compounds: Novell broad-spectrum inh” see also "bitors of RNA virtual effects"). In some embodiments, the compounds described herein can be co-administered with a conventional influenza vaccine.

In some embodiments, a compound described herein (eg, Compound (1) and pharmaceutically acceptable salts thereof, eg, Compound A (1) Form 1 / 2H ₂ O HCl in Form A Salt, Form F compound (1). HCl salt of 3H ₂ O, Form D compound (1) HCl salt, Form A compound (1), Form B compound (1) Hydrate and Form A Compound (1) tosylate salt) can be co-administered with zanamivir. In some embodiments, the compounds described herein can be co-administered with flavipiravir (T-705). In some embodiments, the compounds described herein can be co-administered with oseltamivir. In some embodiments, the compounds described herein can be co-administered with amantadine or rimantadine. Oseltamivir can be administered in the dosing regimen specified on the label. In some specific embodiments, oseltamivir is administered 75 mg twice daily or 150 mg once daily.

  Pharmaceutical composition

  The compounds described herein can be formulated into a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, diluent, adjuvant or vehicle. In one embodiment, the invention relates to a pharmaceutical composition comprising a compound of the invention as described above and a pharmaceutically acceptable carrier, diluent, adjuvant or vehicle. In one embodiment, the present invention is a pharmaceutical composition comprising an effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent, adjuvant or vehicle. is there. Pharmaceutically acceptable carriers include, for example, pharmaceutical diluents, excipients or carriers that are appropriately selected for the intended mode of administration and are consistent with conventional pharmaceutical practice.

  “Effective amount” includes “therapeutically effective amount” and “prophylactically effective amount”. The term “therapeutically effective amount” refers to an amount effective in treating and / or reversing influenza virus infection in a patient infected with influenza. The term “prophylactically effective amount” refers to an amount effective in preventing a pandemic of influenza virus infection and / or in substantially reducing its probability or size. Specific examples of effective amounts are those described above in the section titled Use of the disclosed compounds.

  Pharmaceutically acceptable carriers can contain inert ingredients that do not unduly inhibit the biological activity of the compound. A pharmaceutically acceptable carrier should be biocompatible, eg, non-toxic, non-inflammatory, non-immunogenic, or have other undesirable reactions or side effects when administered to a subject. Should be absent. Standard pharmaceutical formulation techniques can be used.

  A pharmaceutically acceptable carrier, adjuvant or vehicle as used herein is any and all solvents, diluents or other liquid vehicles, dispersion aids suitable for the particular dosage form desired. Agents, suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants and the like. Remington's Pharmaceutical Sciences, Sixteenth Edition, E.M. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutically acceptable compositions and known techniques for preparing them. Has been. Except where any conventional carrier medium is incompatible with the compounds described herein (e.g., by providing any undesirable biological effect or otherwise pharmaceutically acceptable). Its use, by interacting in a deleterious manner with any other component (s) of the composition that can be made, is contemplated to be within the scope of the present invention. As used herein, the phrase “side effects” encompasses unwanted and adverse effects of a treatment (eg, a prophylactic or therapeutic agent). Side effects are not always desired, but unwanted effects are not necessarily harmful. An adverse effect from a treatment (eg, a prophylactic or therapeutic agent) can be harmful, uncomfortable, or dangerous. Side effects include fever, chills, lethargy, gastrointestinal toxicities (including gastric and intestinal ulcers and erosions), nausea, vomiting, neurotoxicity, nephrotoxicities (renal toxicities) (papillary necrosis and chronic Including symptoms such as interstitial nephritis), hepatotoxicity (including increased serum liver enzyme levels), myelotoxicity (including leukopenia, myelosuppression, thrombocytopenia and anemia), dry mouth, gold, pregnancy Include but are not limited to lengthening, weakness, somnolence, pain (including muscle pain, bone pain and headache), hair loss, asthenia, dizziness, extrapyramidal symptoms, inability to sit, cardiovascular disorders and sexual dysfunction .

  Some examples of materials that can function as pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (eg, human serum albumin), buffer substances (eg, twin 80, Phosphate, glycine, sorbic acid or potassium sorbate), partial glyceride mixtures of vegetable saturated fatty acids, water, salts or electrolytes (eg protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride or zinc salts), Colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, polyacrylate, wax, polyethylene-polyoxypropylene-block polymer, methylcellulose, hydroxypropylmethylcellulose, wool fat, sugar (eg, lactose, gluco- Starch (eg corn starch and potato starch); cellulose and its derivatives (eg sodium carboxymethylcellulose, ethylcellulose and cellulose acetate); tragacanth powder; malt; gelatin; talc; excipients (eg cocoa butter and Suppository wax); oils (eg, peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil); glycols; eg (such a) propylene glycol or polyethylene glycol; esters (eg ethyl oleate and Agar; buffer (eg, magnesium hydroxide and aluminum hydroxide); alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol And phosphate buffers, and other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, but are not limited to these, and colorants, releasing agents, Coating agents, sweeteners, flavors and fragrances, preservatives and antioxidants may also be present in the composition according to the judgment of the formulator.

  IV. Administration method

  The compounds and pharmaceutically acceptable compositions described above can be administered to humans and other animals, orally, rectally, parenterally, in the bath, depending on the severity of the infection being treated. In addition, it can be administered intravaginally, intraperitoneally, topically (by powder, ointment or droplets), buccal, as an oral spray or nasal spray.

  Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. Liquid dosage forms contain, in addition to the active compound, inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, Ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oil (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofur It may include furyl alcohol, polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

  Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. A sterile injectable preparation may be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, in 1,3-butanediol It can also be a liquid. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.A. S. P. And isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

  Injectable formulations are made of sterilized solid compositions that can be dissolved or dispersed before use, eg, by filtration through a bacteria-retaining filter, or in sterile water or other sterile injectable medium. It can be sterilized by incorporating a sterilant into the form.

  In order to prolong the effect of the compounds described herein, it is often desirable to delay the absorption of the compound from subcutaneous or intramuscular injection. This can be accomplished by using a liquid suspension of crystalline or amorphous material with poor water solubility. The absorption rate of a compound depends on its dissolution rate, which can further depend on crystal size and crystal form. Alternatively, delayed absorption of a parenterally administered compound form is accomplished by dissolving or suspending the compound in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the compound in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of compound to polymer and the nature of the particular polymer employed, the rate of compound release can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Injectable depot forms are also prepared by entrapping the compound in liposomes or microemulsions that are compatible with body tissues.

  Compositions for rectal or vaginal administration are particularly suitable non-irritating which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity to release the active compound A suppository that can be prepared by mixing a compound described herein with an excipient or carrier of, for example, cocoa butter, polyethylene glycol or a suppository wax.

  Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound comprises at least one inert pharmaceutically acceptable excipient or carrier (eg, sodium citrate or dicalcium phosphate) and / or a) a filler or Bulking agents (eg starch, lactose, sucrose, glucose, mannitol and silicic acid), b) binders (eg carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidinone, sucrose and acacia), c) water retention agents (eg glycerol) D) disintegrants (eg agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain silicates and sodium carbonate), e) dissolution retardants (eg paraffin), f) absorption enhancers (eg four Grade ammonium compound ), G) wetting agents (eg cetyl alcohol and glycerol monostearate), h) absorbents (eg kaolin and bentonite clays), and i) lubricants (eg talc, calcium stearate, magnesium stearate, solids) Polyethylene glycol, sodium lauryl sulfate and mixtures thereof). In the case of capsules, tablets and pills, the dosage forms may also contain buffering agents.

  Similar types of solid compositions can also be used as fillers in soft and hard gelatin capsules using excipients such as lactose or lactose and high molecular weight polyethylene glycols. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may contain opacifiers as needed and either release only the active ingredient (s) in a particular intestinal tract, in a delayed manner as necessary, or the active ingredient (s) ) May be preferentially released. Examples of embedding compositions that can be used include polymeric substances and waxes. Similar types of solid compositions can also be used as fillers in soft and hard gelatin capsules using excipients such as lactose or lactose and high molecular weight polyethylene glycols.

  Effective compounds can also be in microencapsulated form with one or more excipients as described above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, controlled release coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms, the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms also contain additional materials other than inert diluents, such as tableting lubricants and other tableting aids (eg, magnesium stearate and microcrystalline cellulose), as is common practice. May be included. In the case of capsules, tablets and pills, these dosage forms may also contain buffering agents. They may contain opacifiers as needed and either release only the active ingredient (s) in a particular intestinal tract, in a delayed manner as necessary, or the active ingredient (s) ) May be preferentially released. Examples of embedding compositions that can be used include polymeric substances and waxes.

  Dosage forms for topical or transdermal administration of the compounds described herein include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or A patch can be mentioned. Effective components are admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulations, ear drops, and eye drops are also contemplated as being within the scope of this invention. Furthermore, the present invention contemplates the use of transdermal patches with the added benefit of controlled delivery of compounds to the body. Such dosage forms can be made by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by providing a rate controlled membrane or by dispersing the compound in a polymer matrix or gel.

  The compositions described herein were orally, parenterally, by inhalation spray, topically, rectally, nasally, buccal, vaginally or implanted. It can be administered via a reservoir. The term “parenteral” as used herein is subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or Including but not limited to implantation techniques. Specifically, these compositions are administered orally, intraperitoneally, or intravenously.

  Sterile injectable forms of the compositions described herein can be aqueous or oily suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. A sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. possible. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid and glyceride derivatives thereof are useful in the preparation of injectables as well as natural pharmaceutically acceptable oils such as olive oil or castor oil, in particular polyoxyethylated versions thereof. It is. These oily solutions or suspensions are typically used in the formulation of long chain alcohol diluents or dispersants such as carboxymethylcellulose, or pharmaceutically acceptable dosage forms including emulsions and suspensions. Other dispersants may also be included. Other commonly used surfactants (eg, Tweens, Spans and other emulsifiers) or bioavailability enhancements commonly used in the production of pharmaceutically acceptable solid, liquid or other dosage forms Agents can also be used for formulation purposes.

  The pharmaceutical compositions described herein can be administered orally in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. . In the case of tablets for oral use, carriers that are commonly used include, but are not limited to, lactose and corn starch. Lubricants such as magnesium stearate are also usually added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are required for oral use, the active ingredient is admixed with emulsifying and suspending agents. If desired, certain sweetening, flavoring, or coloring agents may also be added.

  Alternatively, the pharmaceutical compositions described herein can be administered in the form of suppositories for rectal administration. These can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.

  The pharmaceutical compositions described herein are topical, particularly when the treatment target includes a region or organ that is readily accessible by topical application (including diseases of the eye, skin, or lower intestinal tract). Can also be administered. Suitable topical formulations are readily prepared for each of these areas or organs.

  Topical application to the lower intestinal tract can be carried out in a rectal suppository formulation (see above) or in a suitable enema formulation. Topical transdermal patches can also be used.

  For topical application, the pharmaceutical composition may be formulated as a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petroleum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical composition can be formulated as a suitable lotion or cream containing the active component suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2 octyldodecanol, benzyl alcohol and water.

  For ophthalmic use, the pharmaceutical composition can be prepared as a micronized suspension in isotonic saline, pH adjusted and sterilized, or specifically, benzylalkonium chloride, etc. Can be formulated as solutions in isotonic saline, adjusted pH and sterilized, with or without preservatives. Alternatively, for ophthalmic use, the pharmaceutical composition may be formulated as an ointment such as petrolatum.

  The pharmaceutical composition may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to procedures well known in the art of pharmaceutical formulation, and include benzyl alcohol or other suitable preservatives, absorption enhancers that improve bioavailability, fluorocarbons and / or other conventional solubilization. Agents or dispersants can be used to prepare a solution in saline.

  The compounds for use in the methods of the invention can be formulated in unit dosage form. The term “unit dosage form” refers to physically discrete units suitable as unit dosages for a subject undergoing treatment, each unit having a predetermined calculated calculated to provide the desired therapeutic effect. An amount of active substance is included, if necessary, with a suitable pharmaceutical carrier. The unit dosage form can be for one of a single dose per day or multiple doses per day (eg, 1 to 4 times per day or more). If multiple doses per day are used, the unit dosage form can be the same or different for each dose.

  V. Example

Example 1: ^XRPD, general method ^{C 13} solid NMR, DSC measurements

  Thermogravimetric analysis (TGA)

  Thermogravimetric analysis (TGA) was performed on a TA Instruments TGA model Q500 Asset Tag V014840. The solid sample was placed in a platinum sample pan and heated from room temperature to 300 ° C. at 10 ° C./min.

  DSC measurement

  Differential scanning calorimetry (DSC) was performed on a TA Instruments DSC Q200 Asset Tag V015553. Approximately 1-2 mg of solid sample was placed in an airtight aluminum DSC pan with a corrugated lid with pinholes. The sample cell was usually heated under a nitrogen purge.

  SSNMR experiment

Solid state nuclear magnetic spectroscopy (SSNMR) spectra were acquired on a Bruker-Biospin 400 MHz Advance III large aperture spectrometer equipped with a Bruker-Biospin 4 mm HFX probe. Samples were packed into 4 mm ZrO ₂ rotors (approximately 70 mg or less depending on sample availability). Typically, a magic angle rotation (MAS) speed of 12.5 kHz was utilized. In order to minimize the effect of frictional heat during rotation, the temperature of the probe head was set to 275K. Proton relaxation times were measured using ¹ H MAS T ₁ saturation recovery relaxation experiments in order to set an appropriate cycle delay for ¹³ C cross-polarization (CP) MAS experiments. In order to maximize the signal-to-noise ratio of the carbon spectrum, the latency of the ¹³ C CPMAS experiment was adjusted to a time that was at least 1.2 times longer than the measured ¹ H T ₁ relaxation time. The CP contact time for ¹³ C CPMAS experiments was set to 2 ms. A CP proton pulse with a linear gradient (from 50% to 100%) was used. The Hartmann-Hahn match was optimized against the external reference sample (glycine). Fluorine spectra were acquired using the proton decoupling MAS setting, and the delayed delay was set to approximately 5 times the measured ¹⁹ F T ₁ relaxation time. Fluorine relaxation time was measured using proton decoupling ¹⁹ F MAS T ₁ saturation recovery relaxation experiment. Both carbon and fluorine spectra were acquired with SPINAL 64 decoupling used at a field strength of approximately 100 kHz. Chemical shifts are referred to an adamantane external standard, and the high field resonance was set at 29.5 ppm.

  Bruker D8 Discover XRPD Experiment Details

  Powder X-ray diffraction (XRPD) pattern using a Bruker D8 Discover diffractometer (Asset Tag V012842) equipped with a sealed tube source and a Hi-Star area detector (Bruker AXS, Madison, Wis.) And obtained at room temperature in reflection mode. The X-ray generator operated at a voltage of 40 kV and a current of 35 mA. The powder sample was placed in an aluminum holder. Two frames were registered each with an exposure time of 120 seconds. Subsequently, the data was integrated over a range of 4.5 ° to 39 ° 2θ with a step size of 0.02 ° and merged into one continuous pattern.

  Example 2: Preparation of 2-MeTHF solvate of Compound (1) and Compound (1)

Compound (1) can be prepared as described in WO2010 / 148197. For example, an amorphous free base compound (1) is prepared according to WO 2010/148197, followed by conventional chiral separation and purification: SCF chiral chromatography using a modulator comprising Et ₂ NH (comprising compound (1) Et ₂ NH salt was obtained), followed by ion exchange resin treatment. The XRPD data is shown in FIG. Alternatively, compound (1) can be produced as a 2-MeTHF solvate by the following procedure:

Preparation of compound 2a (2-amino-3-bromo-5-fluoropyridine)

To a slurry of 2-amino-5-fluoropyridine (6 kg, 53.6 mol) in water (24 L) at 14 ° C. was added 48% hydrobromic acid (18.5 kg, 110 mol) over 10 minutes. The reaction was exothermic and the temperature rose to 24 ° C. The mixture was re-cooled to 12 ° C., then bromine (9 kg, 56.3 mol) was added in 9 portions over 50 minutes (exothermic, maintained at 20 ° C.). The mixture was stirred at 22 ° C. overnight and monitored by ¹ H NMR of quenched aliquots (1 ml 20% K ₂ CO ₃ , 0.3 ml 10% Na ₂ S ₂ O ₃ and 0.7 ml DCM). The mixture was quenched with 5 drops, the organic layer was evaporated and assayed). The mixture was cooled to 10 ° C. and then quenched by the addition of sodium bisulfite (560 g, 5.4 mol) in water (2 L) and further cooled to 0 ° C. This mixture was added to a cold (−4 ° C.) mixture of DCM (18 L) and 5.4 M sodium hydroxide (35 L, 189 mol). About 35 L at the bottom was filtered through a celite pad, resulting in phase separation. The aqueous layer was re-extracted with DCM (10 L). The organic phase was filtered through a pad of 3 kg magnesol while washing with DCM (8 L). The filtrate was evaporated, triturated with hexane and filtered.

  This initial product from all four runs typically contained about 10% SM (starting material), even though the in-process assay showed 97% completion. These were combined, triturated with 50 ° C. hexane (2 L / kg material), then cooled to 15 ° C. and filtered to give compound 2a (30.0 kg, about 95% purity, 149 mol, 67% ). Chromatography (20 kg silica, eluent of 25-50% EtOAc in hexane) of the mother liquor from the first trituration and repurification gave additional compound 2a (4.7 kg, ca. 99% purity, 24 .4 mol, 11%).

Preparation of compound 3a

An inert 400 L reaction vessel was charged with 2a (27.5 kg, 96% purity, 138 mol), Pd (PPh ₃ ) ₄ (1044 g, 0.90 mol) and CuI (165 g, 0.87 mol), and then toluene. (90 kg) was added. The mixture was deoxygenated by three vacuum-nitrogen cycles and then triethylamine (19.0 kg, 188 mol) was added. The mixture was deoxygenated by another vacuum-nitrogen cycle and then TMS-acetylene (16.5 kg, 168 mol) was added. The mixture was heated to 48 ° C. over 23 hours (first exotherm brought the temperature to a maximum of 53 ° C.) and then cooled to 18 ° C. The slurry was filtered through a celite pad and washed with toluene (80 kg). The filtrate was washed with 12% Na ₂ HPO ₄ (75 L) and then filtered through a pad of silica (25 kg) washing with 1: 1 hexane: MTBE (120 L). The filtrate was evaporated to a brown oil and then dissolved in NMP for the next step. Weight of solution of compound 3a -58 kg, about 50 wt%, 138 mol, 100%. ¹ H NMR (CDCl ₃ , 300 MHz): δ 7.90 (s, 1H); 7.33-7.27 (m, 1H); 4.92 (s, NH ₂ ), 0.28 (s, 9H ) Ppm.

Preparation of compound 4a

Potassium tert-butoxide (17.5 kg, 156 mol) and NMP (45 kg) were charged into an inert 400 L reaction vessel. The mixture was heated to 54 ° C., then a solution of compound 3a (29 kg, 138 mol) in NMP (38 kg) was added over 2.75 hours and rinsed with NMP (6 kg) (exothermic, 70 ° C.-77 ° C. Maintained). The reaction was stirred at 74 ° C. for 2 hours, then cooled to 30 ° C. and a solution of tosyl chloride (28.5 kg, 150 mol) in NMP (30 kg) was added over 1.5 hours and rinsed with NMP (4 kg). It is. The reaction was exothermic and the reaction was maintained at 30-43 ° C. The reaction was stirred for 1 hour while cooling to 20 ° C., then water (220 L) was added over 35 minutes (exothermic, maintained at 18 ° C. to 23 ° C.). The mixture was stirred at 20 ° C. for 30 minutes, then filtered and washed with water (100 L). The solid is dissolved from the filter with DCM (250 kg), separated from residual water, and the organic phase is washed with additional DCM (280 kg) while padding of magnetol (15 kg, upper layer) and silica (15 kg, lower layer). Filtered through. The filtrate was concentrated to a thick slurry (approximately 50 L volume), then MTBE (30 kg) was added (final distillate temperature of 51 ° C.) with continued distillation at a constant volume. Further MTBE (10 kg) was added and the slurry was cooled to 15 ° C., filtered and washed with MTBE (40 L) to give compound 4a (19.13 kg, 95% purity, 62.6 mol, 45%). ). A second crop was obtained by partial concentration of the filtrate (2.55 kg, 91% purity, 8.0 mol, 6%). ¹ H NMR (CDCl ₃ , 300 MHz): δ 8.28-8.27 (m, 1H); 8.06-8.02 (m, 2H); 7.77 (d, J = 4.0 Hz) 7.54-7.50 (m, 1H); 7.28-7.26 (m, 2H); 6.56 (d, J = 4.0 Hz, 1H); 2.37 s, 3H) ppm.

Preparation of compound 5a

To a slurry of N-bromosuccinimide (14.16 kg, 79.6 mol) in DCM (30 kg) at 15 ° C. was added compound 4a (19.13 kg, 95% and 2.86 kg, 91% purity) in DCM (115 kg). , 71.6 mol) was added while rinsing with DCM (20 kg). The mixture was stirred at 25 ° C. for 18 hours, then cooled to 9 ° C. and quenched by adding a solution of sodium thiosulfate (400 g) and 50% sodium hydroxide (9.1 kg) in water (130 L). The mixture was warmed to 20 ° C., the layers were separated and the organic phase was washed with 12% brine (40 L). The aqueous layer was sequentially re-extracted with DCM (4 × 50 kg). The organic phases are combined and 40 L is distilled azeotropically with water, and the solution is then washed with a pad of silica (15 kg, lower layer) and magnesol (15 kg, upper layer) while washing with DCM (180 kg). Filtered. The filtrate was concentrated to a thick slurry (approximately 32 L volume) and then hexane (15 kg) was added. Additional hexane (15 kg) was added (final distillate temperature 52 ° C.) while continuing to distill at a constant volume. The slurry was cooled to 16 ° C., filtered and washed with hexane (25 kg) to give compound 5a (25.6 kg, 69.3 mol, 97%). ¹ H NMR (CDCl ₃ , 300 MHz): δ 8.34-8.33 (m, 1H); 8.07 (d, J = 8.2 Hz, 2H); 7.85 (s, 1H); .52-7.49 (m, 1H); 7.32-7.28 (m, 2H); 2.40 (s, 3H) ppm.

Preparation of compound 6a: BEFTAI reaction

In an inert 400 L reaction vessel, compound 5a (25.6 kg, 69.3 mol), bis (pinacolato) diboron (19 kg, 74.8 mol), potassium acetate (19 kg, 194 mol), palladium acetate (156 g, 0.69 mol). ) And triphenylphosphine (564 g, 2.15 mol) followed by dioxane (172 kg) (which had been deoxygenated separately using a vacuum-nitrogen cycle (x3)). The mixture was stirred and deoxygenated using a vacuum-nitrogen cycle (x2) and then heated to 100 ° C for 15 hours. The mixture was cooled to 35 ° C. and then filtered with washing with 30 ° C. THF (75 kg). The filtrate was evaporated and the residue was dissolved in DCM (ca. 90 L). The solution was stirred with 1 kg of carbon and 2 kg of magnetol for 45 minutes and then filtered through a pad of silica (22 kg, lower layer) and magnesol (10 kg, upper layer) while washing with DCM (160 kg). The filtrate was concentrated to a thick slurry (about 40 L volume), then triturated at 35 ° C. and hexane (26 kg) was added. The slurry was cooled to 20 ° C., filtered, washed with a mixture of DCM (5.3 kg) and hexane (15 kg), then washed with hexane (15 kg) and dried on the filter under nitrogen. Compound 6a (23.31 kg, 56.0 mol, 81%) was obtained as a white solid. ¹ H-NMR is consistent with the desired product, HPLC 99.5%, palladium assay 2 ppm. ¹ H NMR (CDCl ₃ , 300 MHz): δ 8.25 (s, 1H); 8.18 (s, 1H); 8.09-8.02 (m, 2H); 7.91-7.83 (M, 1H); 7.30-7.23 (m, 2H); 2.39 (s, 3H); 1.38 (s, 12H) ppm.

Preparation of compounds 8a and 9a

Compound 8a: Anhydrous 7a (24.6 kg, Apex) and quinine (49.2 kg, Buchler) were added to the reaction vessel followed by anhydrous PhMe (795.1 kg). The reaction vessel was then cooled to −16 ° C. and EtOH (anhydrous, 41.4 kg) was added at a rate to maintain the internal temperature of the reaction vessel below −12 ° C. The highest reaction temperature recorded for this experiment was -16 ° C. The reaction mixture was then stirred at −16 ° C. for 16 hours. A sample was removed and filtered. The solid was dried and evaluated by ¹ H-NMR, which showed that no anhydride remained. The contents of the reaction vessel were filtered. The reaction vessel and subsequent wet cake was washed with PhMe (anhydrous, 20 kg). The resulting solid was placed in a box dryer below 45 ° C. for at least 48 hours with N ₂ flowing. In this experiment, the actual temperature was 44 ° C. and the vacuum was −30 in HG. The material was sampled 2.5 days after drying and the material was shown to be 3% PhMe by NMR. After another 8 hours, the amount of PhMe analyzed showed that the same 3% PhMe was present, so drying was stopped. The white solid weighed 57.7 kg, 76% yield. ¹ H-NMR showed conformity with structure and chiral SFC analysis showed material> 99% ee.

Compound 9a: Kinin salt 8a (57.7 kg) and PhMe (250.5 kg, Aldrich ACS grade,> 99.5%) were added to the reaction vessel and the agitator was started. The contents were cooled to below 15 ° C. and treated with 6N HCl (18 kg of H ₂ O was treated with 21.4 kg of concentrated HCl) while maintaining the temperature below 25 ° C. The mixture was stirred for 40 minutes and visually checked to make sure no solid was present. Stirring was stopped, the phases were allowed to settle and the phases were allowed to separate. The aqueous phase was re-extracted with PhMe (160 kg; less commonly used, calculated as 43 kg). However, due to the minimal volume, more PhMe was added for efficient stirring. The organic phases were combined. Sample the organic phase and perform HPLC analysis to ensure that the product is present (as a test for information only).

  The organic phase was cooled to <5 ° C. (0 ° C. to 5 ° C.) and sodium sulfate (anhydrous, 53.1 kg) was added with stirring for 8 hours (12 hours in this case). The contents of the reaction vessel containing the organic phase were passed through a filter containing sodium sulfate (31 kg, anhydrous) and transferred to a clean dry reaction vessel. The reaction vessel was rinsed with PhMe (57.4 kg) and passed through a filter and transferred to reaction vessel 201. The stirrer was started, an additional amount of PhMe (44 kg) was added and the reaction mixture was cooled to -20 ° C. At that temperature, a potassium tert-pentoxide solution in PhMe was added over 2 hours while maintaining the temperature at -15 to -22 ° C. The reaction mixture was sampled after being held at approximately −20 ° C. for an additional 30 minutes. Sampling was performed by removing an aliquot and immediately quenched into 6N HCl. The target ratio here is 96: 4 (trans: cis).

Once the target ratio was achieved, acetic acid (2.8 kg) was charged to the reaction vessel over 6 minutes. The temperature remained at -20 ° C. The temperature was then adjusted to −5 ° C. and 2N aqueous HCl (65.7 kg water treated with 15.4 kg concentrated HCl) was added. The contents were warmed to 5 ° C. + / − 5 ° C. and stirred for 45 minutes, then warmed to 20 ° C. + / − 5 ° C. for 15 minutes with stirring. The stirrer was stopped and the phase was allowed to settle. The aqueous layer was removed (temporarily stored). The organic phase was washed with water (48 kg, drinking), stirred for 15 minutes, allowed to settle (at least 15 minutes), the aqueous layer was removed and added to the aqueous layer. 1/3 of the prepared buffer (7.9 kg NaH ₂ PO ₄ , 1.3 kg Na ₂ HPO ₄ and 143.6 kg water) (about 50 L) was added to the organic phase and stirred for at least 15 minutes . Agitation was stopped and the phases were allowed to separate for at least 15 minutes. The lower layer was discarded. Another portion of buffer (about 50 L) was used to wash the organic layer as previously described. A third wash was performed as described above.

Vacuum distillation of the PhMe phase (150 L) was started at 42 ° C./−13.9 psig and distilled to an oil volume of approximately 20 L. After the volume was substantially reduced, the mixture was transferred to a smaller container to complete the distillation. Heptanes (13.7 kg) were added and the mixture was warmed to 40 +/− 5 ° C. for 30 minutes, then the contents were cooled to 0-5 ° C. over 1.5 hours. The solid was filtered and the reaction vessel was washed with approximately 14 kg cold (0 ° C. to 5 ° C.) heptanes. The solid was dried under vacuum and then placed in an oven below 40 ° C. under house vacuum (−28 psig) until the LOD was <1%. 15.3 kg, 64%, 96% HPLC purity. ¹ H NMR (400 MHz, CDCl ₃ ) δ 11.45 (br.s, 1H), 6.41 (t, J = 7.2 Hz, 1H), 6.25 (t, J = 7.2 Hz) , 1H), 4.18 (m, 2H), 3.27 (m, 1H), 3.03 (m, 1H), 2.95 (m, 1H), 2.77 (m, 1H), 1 .68 (m, 1H), 1.49 (m, 1H), 1.25 (t, J = 7.2 Hz), 1.12 (m, 1H).

Preparation of compound 10a

  In a three-necked flask equipped with a mechanical stirrer, temperature probe, reflux condenser, dropping funnel and nitrogen inlet, compound 9a (145.0 g, 1 eq) and anhydrous toluene (Aldrich, cat # 244511) (1408 g, 1655 ml) were added. It was charged in a nitrogen atmosphere. To the stirred solution was then added triethylamine (Aldrich, cat # 471283) (140 g, 193 ml, 2.14 eq) in small portions over 5 minutes, during which time an exotherm to a maximum temperature of 27 ° C. was observed. Data acquisition by ReactIR was started. The reaction mixture was then heated to 95 ° C. for 70 minutes. Diphenylphosphoryl azide (Aldrich, cat # 178756) (176.2 g; 138.0 ml, 0.99 equiv) was then added in portions via a dropping funnel over a total of 2.25 hours.

After the addition of diphenylphosphoryl azide was complete (the dropping funnel was rinsed with a small amount of toluene), the resulting mixture was heated at 96 ° C. for an additional 50 minutes. A sample of the reaction mixture diluted in toluene was analyzed by GC / MS and showed consumption of diphenylphosphoryl azide. Benzyl alcohol (Aldrich, cat # 108006) (69.9 g, 67.0 ml, 1.0 equiv) was then added via an addition funnel over 5-10 minutes. The resulting mixture was then heated at 97 ° C. overnight (approximately 19 hours). A sample of the reaction mixture diluted in toluene showed product formation by GC / MS (m / e = 330). The reaction mixture was then cooled to 21 ° C., after which water (870 g, 870 ml) was added in small portions (a slight exotherm to a maximum temperature of 22 ° C. was observed). The reaction mixture was first quenched by adding 500 g of water and mechanically stirred for 10 minutes. The mixture was then transferred to a separatory funnel containing the remaining 370 g of water and then stirred manually. After stirring and phase separation, the organic and aqueous layers were separated (aqueous cut at a pH of about 10). The organic layer was then washed with a further portion of water (870 g; 1 × 870 ml). The organic and aqueous layers were separated (aqueous cut at a pH of about 10). The recovered organic phase was then concentrated to dryness under reduced pressure (45 ° C. to 50 ° C. water bath) to give 215 g of crude compound 10a (approximately 190 ml volume). ¹ H NMR and GC / MS were consistent with compound 10a (containing residual toluene and benzyl alcohol).

Preparation of compound 11a

  Preparation of HCl in ethanol: A three-necked flask equipped with a temperature probe, nitrogen inlet and magnetic stirrer was charged with ethanol (1000 ml, 773 g) under a nitrogen atmosphere. The solution was stirred and cooled in a dry ice / acetone bath until an internal temperature of −12 ° C. was reached. Anhydrous HCl (˜80 g, 2.19 mol) was then slowly bubbled into the cooled solution over 2 hours (temperatures of −24 ° C. to −6 ° C. were observed during the addition). After the addition, the solution was transferred to a glass bottle and warmed to ambient temperature. A sample of the solution was submitted for titration and a concentration of 2.6M was obtained. The solution was then stored overnight in a cold room (approximately 5 ° C.).

Hydrogenation / HCl salt formation: palladium on carbon (Pd / C (Aldrich, cat # 330108), 10% dry basis; (50% wet), 13.11 g, compound 10a on a glass insert for a 2 gallon Parr autoclave. Was added under a nitrogen atmosphere and then wetted with ethanol (93 g; 120 ml). A solution of crude compound 10a (212 g, 1 eq) in ethanol (1246 g; 1600 ml) was then added to the glass insert (rinsed a little with ethanol to help transfer). The glass insert was placed in an autoclave and then HCl in ethanol (prepared as described above; 2.6M; 1.04 equivalents based on compound 10a; 223g; 259ml) was added. The autoclave was sealed and then purged with hydrogen (3 × at 20 psi). The hydrogenation then began over 3 hours under an applied pressure of hydrogen gas (15 psi), at which time the hydrogen pressure appeared to be constant. Analysis of an aliquot of the reaction mixture by ¹ H NMR and GC / MS showed consumption of starting material / product formation. The resulting mixture was then filtered through a celite bed (192 g), after which the celite bed was washed with additional ethanol (3 ×; a total of 1176 g of ethanol was used during the wash). The filtrate (green) was then concentrated under reduced pressure (45 ° C. water bath) to about 382 g ((about 435 ml; 2.9 volumes based on the theoretical yield of compound 11a)). Isopropyl acetate (1539 g; 1813 ml (12 volumes based on the theoretical yield of compound 11a)) was then added to the remainder. The resulting solution was distilled under vacuum while gradually raising the temperature.

The distillation was stopped, after which the remaining solution (370 g, about 365 ml total volume; brownish color) was allowed to stand at ambient temperature over the weekend. The mixture was filtered (isopropyl acetate was used to aid filtration) and the collected solid was washed with additional isopropyl acetate (2 × 116 ml; each wash was approximately 100 g). The solid was then dried under vacuum at 40 ° C. (maximum temperature observed was 42 ° C.) overnight to give 118 g (78.1% over 2 steps) of compound 11a. The ¹ H NMR of the material was consistent with the structure of compound 11a and GC / MS showed 99% purity.

Preparation of compound 13a

Procedure A: A mixture of 5-fluoro-2,4-dichloropyrimidine (12a, 39.3 g, 235 mmol, 1.1 eq) and HCl amine salt (11a, 50 g, 214 mmol) was added with CH ₂ Cl ₂ (169 mL). And the mixture was warmed to 30 ° C. The mixture was then slowly treated with DIEA (60.8 g, 82 mL, 471 mmol, 2.2 eq) via syringe pump for 3 hours. The peak temperature was up to 32 ° C. The reaction was stirred for 20 hours when the reaction mixture was judged complete by HPLC and cooled to room temperature. The resulting reaction mixture was washed sequentially with water (211 mL, pH = 8-9), 5% NaHSO ₄ (211 mL, pH = 1-2), then 5% NaCl aqueous solution (211 mL, pH = 5-6). did.

The organic phase was then distilled under reduced pressure to 190 mL. PhMe was charged (422 mL) and the temperature was set between 70 ° C. and 80 ° C. and the internal temperature between 60 ° C. and 65 ° C. until the volume returned to 190 mL. The mixture was cooled to 37 ° C. with stirring and after about 10 minutes crystallization began to occur and the temperature was observed to rise to 41 ° C. After equilibrating at 37 ° C., the suspension was charged with n-heptane (421 mL) over 3.5 hours and then cooled to 22 ° C. over 1 hour. The mixture was stirred at that temperature overnight and then filtered. The resulting solid on the filter was washed with 10% PhMe in n-heptane solution (2 × 210 mL). The solid was then dried in an oven under vacuum overnight at 50 ° C. while purging with N ₂ . The resulting solid weighed 62 g (88% yield).

  Procedure B: Compound 11a (51.2 g) and Compound 12a (40.2 g) were charged under a nitrogen atmosphere into a three-necked flask equipped with a mechanical stirrer, temperature probe, reflux condenser, nitrogen inlet and dropping funnel. . Dichloromethane (173 ml, 230 g) was added and the resulting mixture was stirred while warming to an internal temperature of 30 ° C. N, N-diisopropylethylamine (85 ml, 63.09 g) was then slowly added via an addition funnel over a period of 2.5-3 hours during which time an exotherm to the observed maximum temperature of 33.5 ° C. Observed. After the addition was complete, the resulting solution was stirred at 30-31 ° C. overnight under a nitrogen atmosphere (approximately 19 hours).

A 100 μl sample of the reaction mixture was diluted with dichloromethane to a total volume of 10 ml and the solution was mixed well. Analysis of the diluted aliquot sample by GC / MS showed that the reaction was complete by GC / MS; product formation was observed (m / e = 328)). The reaction mixture was cooled to 26 ° C. and transferred to a separatory funnel (with the aid of dichloromethane). The mixture was then washed with water (211 ml, 211 g; the pH of the aqueous cut was about 8; a small rag layer was transferred with the aqueous cut), 5% NaHSO ₄ aqueous solution ((50 g of sodium bisulfate 211 ml, 216 g (prepared with monohydrate (Aldrich cat. # 233714) and 950 g water); aqueous cut pH was about 2), then 5% NaCl aqueous solution ((50 g sodium chloride ( Aldrich cat. # S9888) and 950 g of water) (211 ml, 215 g; pH of aqueous cut was about 4-5). The recovered organic phase is then concentrated under reduced pressure (35 ° C. water bath) to approximately 190 ml (2.7 volumes based on the theoretical yield of compound 13a), followed by toluene (Aldrich cat. # 179418). , 422 ml, 361 g). The resulting mixture was concentrated under reduced pressure (55 ° C. to 65 ° C. water bath) to approximately 190 ml (2.7 volumes based on the theoretical yield of compound 13a). Analysis of a sample of the solution at this stage by ¹ H NMR indicated the absence of dichloromethane. The remaining mixture was cooled to 37 ° C. (using a 37 ° C. water bath with stirring in a rotovap). Obvious crystallization was observed during this time. The mixture was then mechanically stirred and heated to approximately 37 ° C. (external heat source set at 38 ° C.), after which n-heptane (430 ml, 288 g; Aldrich cat # H2198) was added to the addition funnel over 3 hours. Slowly added by. After the addition, heating was stopped and the resulting slurry was mechanically stirred overnight while cooling to ambient temperature. The resulting mixture was then filtered and the recovered solid was washed with 10% toluene in n-heptane (2 × 210 ml; each wash was 21 ml (16 g) toluene and 189 ml (132 g) n. -Prepared by mixing heptane). Vacuum was applied until only a little filtrate was observed. The solid was then dried under nitrogen at 50 ° C. to a constant weight (3.5 hours) under a stream of nitrogen, yielding 64.7 g (90%) of compound 13a. Analysis of the solid sample by ¹ H NMR indicated that the material was consistent with the structure, and LC analysis indicated a purity of 99.8% using the provided LC method.

Preparation of compound 14a

  Ethyl ester 13a (85 g, 259 mmol) was dissolved in THF (340 mL) and treated with a solution of LiOH (2M, 389 mL, 778 mmol) for 10 min (temperature of 21-24 ° C.). The mixture was warmed to 45 ° C. with stirring over 17 hours, at which point the reaction was judged complete by HPLC (no SM was observed). The reaction mixture was cooled to room temperature and CH2Cl2 was added (425 mL). A solution of citric acid (2M, 400 mL) was then added slowly over 45 minutes (temperature to 26 ° C.). Note that some white solid formed during charging but dissolved rapidly by stirring. The reaction mixture was stirred for an additional 15 minutes before the phases were separated. After splitting the phases, the pH of the aqueous phase was measured as pH = 4.0. The organic phase was washed with water (255 mL) (stirring for 15 minutes) and the phases were separated. The lower layer (organic layer) containing the desired product was then stored in the refrigerator overnight.

  The organic phase was concentrated under reduced pressure until approximately 150 mL (estimated 1.76 vol for SM) (pot set at 65 ° C.). IPA (510 mL) was added and distilled under reduced pressure to 255 mL (3 vol) (cooler temperature setting of 85 ° C.). The level of solvent was brought to approximately 553 mL (6.5 vol) by adding IPA (298 mL). Then, water (16 mL) was added and the reaction mixture was heated to reflux (77 ° C.) with sufficient stirring to dissolve the solid precipitated on the vessel wall. The reaction mixture was then slowly cooled to 65 ° C. (over 60 minutes) and kept in place (all material was still in solution (sampled for residual solvent analysis)). When the reaction was further cooled to 60 ° C., the reaction mixture appeared slightly opaque. After stirring for 15 minutes, the mixture was further cooled to 55 ° C. More product precipitated, but the mixture is still dilute and easily stirred. Water (808 mL) was added very slowly (2.5-3 hours) while maintaining the temperature at approximately 55 ° C. The mixture was then cooled to 22 ° C. over 2 hours and stirred overnight. The material was then filtered and washed with a mixture of water: IPA (75:25, 2 × 255 mL). The acid was dried overnight in a 55 ° C. vacuum oven. 69 g of acid 14a was obtained as a white solid in 88% yield. The material was analyzed by HPLC to be> 99% pure.

Preparation of compound 15a: Suzuki coupling

14a (91.4 g, 305 mmol), 6a (158.6 g, 381 mmol, 1.25 equivalent), Pd (OAc) ₂ (0.34 g, 1.5 mmol, 0.5 mol%), X-Phos (1.45 g) , 3.0 mmol, 1.0 mol%) and K ₂ CO ₃ (168.6 g, 1220 mmol, 4 eq) were added THF (731 mL, 8 vol) and water (29 mL, 0.32 vol). The reaction mixture was sparged with N ₂ for 30 minutes, then warmed to 65-70 ° C. and stirred for 5 hours. HPLC analysis of the reaction mixture showed 99.3% conversion. The reaction mixture was cooled to 22-25 ° C. and water was added. The mixture was stirred, the phases were separated, and the aqueous phase was decanted. An aqueous solution of 18 wt% NaCl (half-saturated aqueous NaCl solution) was added to the organic phase and the pH of the mixture was adjusted to 6.0-6.5 using 2N HCl. The phases were separated and the aqueous phase was decanted. The organic phase was concentrated to a minimum volume and acetonitrile was added. This process was repeated once more and the final volume was brought to 910 mL (10 vol) by adding acetonitrile. The slurry was warmed to 80-85 ° C. over 6 hours and then cooled to 20-25 ° C. The slurry was stirred for 2 hours and then filtered. The solid was rinsed with acetonitrile to give 15a (161 g, 89% yield).

Preparation of compound (1): detosylation step

  To 15a (25 g, 45.2 mmol) was added THF (125 ml, 5 vol) followed by MP-TMT resin (6.25 g, 25 wt%). The mixture was stirred at 20-25 ° C. for 16 hours and filtered while rinsing with 1 vol of THF. This resin treatment process and filtration were repeated. The THF solution was concentrated to 5 vol. To the mixture at 22 ° C. to 25 ° C. was added an aqueous solution of 2M LiOH (90.3 mL, 4 eq). The reaction mixture was warmed to 40 ° C. to 45 ° C. and stirred for 5 hours. HPLC analysis showed 99.7% conversion. The reaction mixture was cooled to 22-25 ° C. and MTBE (50 mL, 2 vol) was added. Phase separation occurred. The lower aqueous phase was collected. The aqueous phase was extracted with MTBE. The lower aqueous phase was collected. To the aqueous phase was added 2-MeTHF and the mixture was stirred. The pH of the mixture was adjusted to 6.0-6.5 and the lower aqueous phase was decanted. The organic phase was washed with pH 6.5 buffer. The organic phase was concentrated to 85 mL, diluted with 2-MeTHF (150 mL) and concentrated to a final volume of 180 mL. The resulting slurry was warmed to 70 ° C. to 75 ° C., stirred until completely dissolved, and then cooled to 45 ° C. to 50 ° C. to obtain a slurry. The slurry was stirred for 1 hour and then heptane (180 mL) was added. The slurry was cooled to 20-25 ° C. over 1 hour and stirred for 16 hours. The batch was filtered while rinsing the solid with heptane. The solid was dried to obtain a crude compound (1) .2-MeTHF solvate. 79% yield.

  Example 3: Formation of HCl salt polymorph of compound (1)

3A: Preparation of Form A Compound (1), HCl salt of 1 / 2H ₂ O

A HCl salt of Form A Compound (1) .1 / 2H ₂ O is converted to 2-methyltetrahydrofuran (2-MeTHF) solvate of Compound (1) in a mixture of water and organic solvent (s) ( 1 equivalent) (compound (1) · 1 · (2-MeTHF)) with hydrogen chloride (wherein the mixture of water and organic solvent (s) is 0.05 to Had a water activity of 0.85). The specific reaction conditions used are summarized in Table 2 below.

Alternatively, the HCl salt of Form A Compound (1) .1 / 2H ₂ O was also prepared by the following procedure: Procedure A: Compound (1) .2-MeTHF (953 g, 2.39 mol) in 30 L jacket The reaction vessel was charged with IPA (15 L) and water (0.57 L). The stirrer was started and the reaction mixture was warmed to 73 ° C to bring everything into solution and then cooled to 50 ° C to 55 ° C. The reaction mixture was treated at 50-55 ° C. with freshly prepared HCl in IPA (0.83 M, 4.34 L) by slow addition over 4 hours. It should be noted that the mixture becomes thick after about half the time. The reaction was sampled to confirm by XRPD that it was in the correct form. After the addition, the chiller was programmed to drop to 0 ° C. over 480 minutes with stirring. After confirmation of morphology by XRPD analysis, the slurry was filtered with two filters. The reaction vessel was washed with 3 L IPA and each filter cake was washed with about 1.5 L IPA of IPA wash from the reaction vessel. The cakes were air dried overnight by suction. The cakes were then placed in a box dryer for 24 hours with no heating and purging with N ₂ under vacuum (22 in Hg). From residual though analysis of solvent and water, IPA of 505ppm, 2-Me-THF and approximately 2.15% _{H 2} O of 8ppm showed. The material was removed from the oven and ground together to break up lumps, yielding 805 g of compound (1) · 1 / 2H ₂ O HCl salt. Procedure B: Alternatively, acetone instead of IPA was used in a manner similar to that described in Procedure A above to form the HCl salt of Compound (1) .1 / 2H ₂ O.

XRPD and C ¹³ SSNMR data of the HCl salt of Form A compound (1) .1 / 2H ₂ O are shown in FIGS. 1 and 2, respectively. Certain XRPD peaks and C ¹³ SSNMR peaks observed are summarized in Table 2 and Table 3, respectively.

The prepared HCl salt of Form A compound (1) .1 / 2H ₂ O was found to be stable in the following solvent systems including, but not limited to: chlorobenzene, cyclohexane, 1,2 -Dichloroethane, dichloromethane, 1,2-dimethoxyethane, hexane, 2-methoxyethanol, methylbutylketone, methylcyclohexane, nitromethane, tetralin, xylene, toluene, 1,1,2-trichloroethane, acetone, anisole, 1-butanol, 2-butanol, butyl acetate, t-butyl methyl ether, cumene, ethanol, ethyl acetate, ethyl ether, ethyl formate, heptane, isobutyl acetate, isopropyl acetate, methyl acetate, 3-methyl-1-butanol, methyl ethyl ketone, 2-methyl -1-propanol, penta , 1-propanol, 1-pentanol, 2-propanol, propyl acetate, tetrahydrofuran, methyl tetrahydrofuran.

Specifically, a sample of the compound was loaded into a 2 mL HPLC vial with 500 μl of solvent for solubility testing and stability testing of Form A compound (1) · 1 / 2H ₂ O in HCl salt. The mixture was stirred at ambient temperature for 2 weeks and then filtered through a centrifuge. The resulting solid was analyzed by XRPD and the solution was analyzed for solubility by quantitative NMR against a hydroquinone standard. The results are summarized in Table 4.

  Thermogram data was obtained by placing the sample in a platinum sample pan and heating from room temperature to 300 ° C. at 10 ° C./min (data not shown). The thermogram data showed a 2.1% weight loss from 30 ° to 170 ° C., consistent with the theoretical hemihydrate (2.0%).

  DSC thermogram data was obtained by heating the sample from room temperature to 300 ° C. at 10 ° C./min (data not shown). The DSC thermogram showed a melting / decomposition onset temperature of 200 ° C. to 260 ° C. after a dehydration onset temperature of 50 ° C. to 100 ° C.

3B: Preparation of Form F Compound (1) 3H ₂ O HCl Salt

The HCl salt of Form F compound (1) · 3H ₂ O is obtained by slurrying Form A Compound (1) · 1 / 2H ₂ O HCl salt in isopropanol and water, or acetone and water, or water. Can be prepared (with a water activity value equal to or greater than 0.9).

For example, a slurry of 100 mg Form A compound (1) .1 / 2H ₂ O HCl salt in 5 mL isopropanol / water or acetone / water with a water activity of 0.9 was stirred overnight at ambient temperature. The supernatant was decanted, and the solid material obtained was gently air dried to obtain the HCl salt of Form F compound (1) · 3H ₂ O.

XRPD and C ¹³ SSNMR data of the HCl salt of Form F compound (1) .3H ₂ O are shown in FIGS. 3 and 4, respectively. Certain XRPD peaks and C ¹³ SSNMR peaks observed are summarized in Table 5 and Table 6, respectively.

  MDSC thermograms were obtained by heating the sample at -20 ° C. to 350 ° C. at 2 ° C./min (data not shown) and adjusted at ± 1 ° C. every 60 seconds. The MDSC thermogram showed dehydration below 150 ° C., melting and recrystallization between 150 ° C. and 200 ° C., and degradation above 250 ° C.

  The form of thermogravimetric analysis (TGA) was also performed. The thermogram showed a 12% weight loss to 125 ° C., which was close to the theoretical trihydrate (11%). The second step weight loss below 200 ° C. was shown to be a decrease in HCl by TGA-MS. The onset of melting / decomposition was approximately 270-290 ° C.

  3C: Preparation of HCl salt of compound (1) in form D

The HCl salt of anhydrous form D compound (1) can usually be made by dehydrating the HCl salt of form A compound (1) .1 / 2H ₂ O. This dehydration could be done by heating or purging with dry nitrogen, or a combination of the two. For example, heating the HCl salt of ₂ mg of Form A Compound (1) .1 / 2H ₂ O HCl on a hot plate produced the desired anhydrous Form D at approximately 85 ° C.

XRPD and C ¹³ SSNMR data of the HCl salt of anhydrous form D (1) are shown in FIGS. 5 and 6, respectively. Certain XRPD peaks and C ¹³ SSNMR peaks observed are summarized in Table 7 and Table 8, respectively.

  3D: Water activity test

In the water activity of 0.0 to 0.8 of isopropyl alcohol / water, the compound of Form A (1) • 1 / 2H ₂ O of seeded with HCl salt of Form F (1) • 3H ₂ O Competitive slurry studies of HCl salt show that after stirring for approximately 2 weeks under ambient conditions, Form A is the anhydrous HCl salt of Form D (1), Form F (1) .3H ₂ O HCl and It was shown to be the most stable form of the HCl salt of Form A compound (1) .1 / 2H ₂ O. Form A (1) .1 / 2H ₂ O HCl salt of Form A was converted to Form F (1) .3H ₂ O HCl salt at an IPA / water activity of 0.9. The results of these studies are summarized in Table 9 below.

Anhydrous HCl salt of Form D (1) (“D Form”), Form F Compound (1). HCl salt of 3H ₂ O (“F Form”) and Form A Compound (1) .1 / 2H A phase diagram of temperature versus water activity for the transition between ₂ O HCl salts ("Form A") is shown in FIG.

  3F: Amorphous HCl salt of compound (1)

Treating the amorphous HCl salt of compound (1) with water and Me ₂ NEt salt (1.985 g) of compound (1) in 2-MeTHF with 1.05 equivalents of NaOH followed by HCl. To remove the amine and crush out from the aqueous layer (pH 2-3). All organic phases were removed by concentrating the resulting slurry and then filtered. The resulting solid was rinsed with a small amount of water and dried. The Me ₂ NEt salt of compound (1) was prepared according to WO 2010/148197, followed by conventional chiral separation and purification: SCF chiral chromatography using a modifier containing Me ₂ NEt (this gave the compound (1) Me ₂ NEt salt was produced).

  Example 4: Formation of polymorph of free base compound (1)

  4A: Preparation of form A free base compound (1)

  Form A free base compound (1) was produced by the following procedure: Crude amorphous free base compound (1) (approximately 135 g) was transferred to a 4 L jacketed reaction vessel and ethanol ( 2.67 L) and water (0.325 L) were added (10% aqueous solution). The mixture was heated to reflux. A 20% aqueous solution was prepared by adding water (300 mL) to the resulting mixture of step 2). The resulting mixture was then cooled to 55 ° C. (rate = −1 ° C./min) and subsequently held for 30 minutes. Then, seed crystals of free base Form A compound (1) (1.5 g, 3.756 mmol) were added to the cooled mixture and the resulting mixture was held for 30 minutes when the product precipitated. A seed crystal of crystalline free base Form A compound (1) was made by slurrying amorphous free base compound (1) (20 mg) in nitromethane (0.5 mL). Additional seed material of crystalline free base Form A compound (1) was prepared by adding amorphous free base compound (1) (900 mg) in acetonitrile (10 mL) with seed crystals obtained using nitromethane. It was prepared by slurrying. A 40% aqueous solution was prepared by slowly adding water (795.0 mL) to a mixture containing crystalline free base Form A compound (1) seed crystals. The resulting mixture was slowly cooled to 0 ° C. (about −10 ° C./hour) and subsequently held for 2 hours. The solid material was then filtered, air dried and then dried in an oven at 60 ° C. for an additional 18 hours.

  Alternatively, the 2-methyl THF solvate of the free base compound (1) is used in place of the amorphous free base compound (1) and the A form is prepared in a similar manner as described above. The free base compound (1) was obtained.

  The prepared Form A compound (1) was found to be stable in the following solvent systems including, but not limited to: acetonitrile, chlorobenzene, chloroform, cyclohexane, 1,2-dichloroethane, dichloromethane, 1,2-dimethoxyethane, ethylene glycol, formamide, hexane, methylbutylketone, methylcyclohexane, N-methylpyrrolidinone, nitromethane, tetralin, toluene, 1,1,2-trichloroethane, acetic acid, anisole, 1-butanol, butyl acetate , Cumene, ethyl acetate, ethyl ether, ethyl formate, heptane, isobutyl acetate, isopropyl acetate, 3-methyl-1-butanol, 2-methyl-1-propanol, pentane, propyl acetate, water, water-isopropanol (1: 3 vol / Vo ) And water - acetonitrile (1: 1vol / vol; 1: 3vol / vol).

XRPD and C ¹³ SSNMR data of Form A compound (1) are shown in FIGS. 7 and 8, respectively. Certain observed XRPD peaks and C ¹³ SSNMR peaks are summarized in Table 10 and Table 11, respectively.

  In a TA Instruments TGA model Q500, a sample of Form A compound (1), a product, is placed in a platinum sample pan, followed by heating the pan from room temperature to 300 ° C. at 10 ° C./min. Was subjected to thermogravimetric analysis (data not shown here). The thermogram showed that the onset of decomposition was approximately 293 ° C.

  A DSC thermogram for Form A compound (1) was also obtained using TA Instruments DSC Q200. The sample in that form was heated to 350 ° C. at 10 ° C./min. The DSC thermogram showed that the melting temperature was approximately 278 ° C.

  4B: Preparation of hydrate of free base compound (1) in form B

  The hydrated form of the free base compound (1) is of the same form as the A-form free base compound (1), that is, the A-form free base compound (1) is hydrated when exposed to high humidity. Can be freely converted to the B shape, and can return to its original state when the humidity drops. According to the phase change measured using DSC experiments (data not shown), the transition temperature is close to ambient temperature and varies with water activity. For example, a hydrate form was observed when the water activity was greater than 0.6 (eg, 0.6-1.0) at ambient temperature.

4C: Preparation of amorphous free base compound (1)

Suzuki coupling was performed by dissolving chloropyrimidine, compound 13a, boronate ester compound 6a, catalyst Pd (OAc) ₂ and ligand (X-Phos) in 10 vol 2-MeTHF. The mixture was heated to 65 ° C. and 2 vol of a 50% aqueous solution of K ₃ PO ₄ was added at a rate to maintain the reaction mixture at 65 ° C. Both reactions proceeded to complete conversion, then cooled to 20 ° C. and filtered through celite. The aqueous layer was separated and discarded, and the organic layer was washed with 5% aqueous NaCl solution and then concentrated to dryness to give approximately 3.5 kg of dark green paste for each. The crude oil was divided into four equal parts, slurried with 400 g SiO ₂ and 500 g florisil, and passed through a 2.3 kg SiO ₂ column with heptane / EtOAc (5: 1-3: 1, 2 L fractions). All fractions containing product were combined, eluting at These fractions were concentrated to dryness to obtain approximately 2.9 kg of compound 21a.

Compound 21a was dissolved in 10 vol (25 L) CH ₃ CN and treated with 4 equivalents of HCl at 70 ° C. (4.31 L of 4N HCl in 1,4-dioxane) for 15 hours. The reaction was judged 100% complete by HPLC and the dilute slurry was cooled to 20 ° C. for 1 hour. TBME (28 L, 11 vol) was added at 0.5 L / min, and at the end of the addition, the slurry became very thick (gelatinous). After stirring for 4-5 hours, the slurry became thinner. The resulting solid was collected by suction filtration and washed with 3 × 5 L TBME to obtain a low density cake, which was dried for 3 days under N ₂ vapor for 1.71 kg (86% yield). , 98.9% AUC purity).

  A solution of NaOH (55.60 mL of 2M, 111.2 mmol) was added to a suspension of compound 22a.HCl (10 g, 22.23 mmol) in 2-MeTHF (100.00 mL) at 20 ° C. The reaction mixture was stirred at 60 ° C. for 5 hours and then further stirred at 67 ° C. After stirring for about 22 hours, 100 mL (10 vol) of 2-MeTHF was added to the resulting mixture. The batch was then cooled to 0 ° C. The crude free base compound (1) was obtained by adjusting HCl to pH 6.6 by adding HCl to the obtained mixture. The crude material in 60 mL (6 vol) 2-Me-THF was heated to 50 ° C. To the resulting mixture, 50 mL (5 vol) of n-heptane was added over 1 hour. The batch was then cooled to 20 ° C. The solid product was filtered and the solid product was further purified by column chromatography (EtOAc / heptane 2: 1 to 4: 1). The XRPD data showed an amorphous free base compound (1).

  Alternatively, the amorphous free base compound (1) is a form A free base compound (1) and a solvent selected from 2-ethoxyethanol, 2-methoxyethanol, t-butyl methyl ether, formic acid or methyl ethyl ketone. From a mixture (stirred at ambient temperature) (see, eg, Table 13 below).

  4D: Preparation of 2-MeTHF solvate of free base compound (1)

  Compound (1) .1 (2-MeTHF) was prepared as described in Example 2 above. The XRPD data is shown in FIG. The particular XRPD peaks observed are summarized in Table 12.

  4F: Solubility and stability data of Form A free base compound (1) and amorphous compound (1) in various solvent systems

  The solubility and stability of form A free base compound (1) ("A form") and amorphous compound (1) ("amorphous") in various solvent systems were compared to form A compound (1). Were tested at ambient temperature in a manner similar to that described above for the solubility and stability of the HCl salt. The data obtained is summarized in Table 13.

  Example 5: Preparation of tosylate salt of Form A compound (1)

  The tosylate salt of Form A compound (1) was prepared by slurrying the amorphous free base compound (1) (500 mg) and p-toluenesulfonic acid in acetonitrile (20 ml). The sample was stirred overnight. The XRPD data is shown in FIG. The particular XRPD peaks observed are summarized in Table 14.

  Alternatively, by using 2-methyl THF solvate of the free base compound (1) instead of the amorphous free base compound (1), the compound of form A in a manner similar to that described above The tosylate salt (1) could be prepared.

  Example 6: Preparation of compound (1)

  A. Compound (1) tablets

  Composition

Form A compound (1). HCl salt of 1/2 H ₂ O (hereinafter simply, compound (1) in Example 6) was used for tablet formation. All excipients were purchased from approved suppliers according to the current monograph of the European Pharmacopeia and USP / NF.

  The blend composition and batch size for the pre-granulation blend and granulation binder solution are shown in Table 15A. The binder solution batch size contained over 100% for pump calibration and solution line priming. The theoretical composition of the compression blend is also shown in Table 15A. The actual amount for the batch was calculated based on the dry granule yield. The composition of the film coating suspension and the approximate batch size are shown in Table 15B, which contained over 100% for pump calibration and suspension line priming. The target amount for film coating was 3.0% w / w of tablet weight.

  Preparation of binder solution

  The binder solution consisted of Povidone and water. The solution was prepared based on 40% water content in the final granulation. Therefore, the total amount of solids in the solution (Povidone) was 3.6% (w / w). A 100% excess was prepared, such as for priming the line. Based on visual inspection of the start of the granulation run, an additional stock solution (38-42%) of +/- 2% water was prepared in the final granulation. Typically, 87.00 g of Povidone K30 and 2320.00 g of purified (DI) water were weighed and Povidone K30 was added to a vessel containing DI water with constant stirring. After the addition, the vessel was sealed to minimize evaporation and the solution was stirred until all solids present were completely dissolved.

  Flow of wet granulation process

Wet granulation was performed according to the procedure described below: Excess (10%) amounts of compound (1), Avicel PH-101, Fastflo lactose and croscarmellose sodium were weighed (see Table 15A) ). They were screened using a cone mill equipped with a 20 mesh hand screen or an 813 μm grated mesh screen at 1000 rpm (for U5 Quadro Co-mill). The screened material was placed in a separate bag or container. The materials were then transferred to a blender and blended for 15 minutes, usually 15 RPM. The blended material was ground using a U5 Quadro cone mill equipped with a 4 mm square hole screen at 1000 rpm. The blending process was repeated to re-blend the milled material. The reblended material was then fed into a twin screw granulator. The bulk wet granulation was fed to the granulator using a loss-in-weight feeder (K-tron or similar). The resulting material was then granulated. Binder fluid (see Table 15A) was injected into the twin screw granulator using a peristaltic pump. The ratio of the solution feed rate to the powder feed rate was 0.4095. For example, if the powder feed rate was 15.00 g / min, the solution feed rate was 0.4095 ^* 15.00 = 6.14 g / min (where the water content was 40% (Based on dry mass)). The granule sub-batch was collected in a tared drying tray. The recovered material was sprayed uniformly onto the tray and the material was dried in an oven to form dry granules. The dried granules were continuously fed into a corn mill by placing them in a K-tron followed by grinding.

  Extragranular blending and compression process

  The extragranular blending and compression process was carried out by the procedure described below: The amount of extra-granular excipients based on the compressed blend composition was weighed out. U5 Comil was used with a 32C screen and a round bar impeller at 1000 rpm to screen out the weighed excipients. First, the comminuted granules of compound (1) were added to a blender containing sieved Avicel PH-102 and Ac-Di-Sol. They were blended for 8 minutes at 16 RPM. Sodium stearyl (SSF) was sieved through a mesh 50 hand screen and placed in a suitable container. A portion of the extragranular blend equal to approximately 10 times the mass basis of the amount of SSF was placed in the container with the SSF, bag blended for 30 seconds, and then the mixture was added to the bin blender. . All materials were then blended for 2 minutes at 16 rpm. The final blend was then compressed according to specified tablet compression process parameters.

  Film coating process

  Film coating was applied to the core tablets in a Vector VPC 1355 pan coater as a 15% w / w Opadry II white # 33G aqueous suspension. The target coating was 3.0% w / w of the core tablet weight and the acceptable range was 2.5% to 3.5%. To achieve this, an amount of coating suspension equal to 3.2% weight gain was sprayed, resulting in a coating of 3.0% assuming a coating efficiency of 95%.

  Intravenous (IV) formulation of compound (1)

Form A Compound (1) · HCI salt of 1 / 2H ₂ O (hereinafter briefly, Compound (1) in Example 6) as a 2 mg / mL solution for intravenous (IV) administration Supplied. The composition of the solution was provided in Tables 16A and 16B, with quality references and the function of each component.

  Example 7: In vivo assay for combined use of compound (1) with or without oseltamivir

Infected mice are combined with a clinically relevant dose of oseltamivir starting 48 hours after the influenza A challenge or 2 hours before the influenza B challenge, and vehicle or increasing dose levels of Form A compound (1) 1 / treated with 2H ₂ O of the HCl salt.

  Methods: In these studies, the HCl salt of Form A Compound (1) hemihydrate (hereinafter, briefly referred to as Compound (1) in Example 7), 0.5% (w / v ) Formulated in a vehicle containing MC (Sigma-Aldrich, St Louis, Mo.) to obtain a uniform suspension. Compound doses were based on the HCl salt of Compound (1) hemihydrate. Oseltamivir was formulated in distilled deionized water to obtain a uniform suspension. The combination of compound (1) and oseltamivir was formulated in a vehicle containing 0.5% (w / v) MC. The combination formulation was prepared at the beginning of each study and stored at 4 ° C. for up to 10 days with stirring in the dark. All formulations and vehicles were administered to mice by oral gavage at a dose volume of 10 mL / kg.

  Male Balb / c mice (5-7 weeks old, 17-19 grams) are anesthetized and inoculated with a lethal dose of mouse adapted influenza virus A / PR / 8/34 or B / Mass / 3/66 by intranasal instillation. did. Eight mice were included per study group. Treatment was initiated +48 hours after inoculation for influenza A or 2 hours before inoculation for influenza B. In the influenza A study, vehicle (10 mL / kg) and 0.1-10 mg / kg dose of Compound (1) were administered orally (PO) alone or 10 mg / kg twice daily (BID) for 10 days. It was administered in combination with kg oseltamivir. In the influenza B study, vehicle (10 mL / kg) and 1-10 mg / kg dose of Compound (1) were administered twice daily (BID) for 10 days (PO), alone or 10 mg / kg oseltamivir. And was administered in combination. Mice were weighed and observed daily for signs of morbidity for 21 days after infection. In addition, lung function was monitored by unrestricted WBP (Buxco, Troy, NY).

Influenza A / PR / 8/34 (VR-1469) and influenza B / Mass / 3/66 (VR-523) were obtained from ATCC (Manassas, VA). Stocks were prepared by standard methods known in the art. Briefly, the virus is sub-inoculated into Madin-Darby canine kidney cells (MDCK cells, CCL-34, ATCC) with low infection efficiency, and the supernatant is collected after approximately 48 hours and centrifuged at 650 × g for 10 minutes. did. Virus stocks were frozen at −80 ° C. until use. Serial dilutions of virus samples, infection of replicated MDCK cultures, and 96 hours later cytopathic effect (CPE) was measured based on ATP content (CellTiter-Glo, Promega, Madison WI), followed by virus titer ( TCID ₅₀ / ml) was calculated by the Spearman-Karger method.

  Mice were weighed daily for 21 days after infection. To compare groups, body weight data were analyzed using a two-way ANOVA and Bonferroni post-test. P values less than 0.05 were considered significant.

  Mice were observed daily for 21 days after influenza infection. Any mouse scored positive for 4 of the following 6 observations (> 35% weight loss, disturbed hair, rounded back posture, respiratory distress, decreased mobility or hypothermia) Was euthanized according to the guidelines established by the Vertex Institutional Animal Care and Use Committee and scored dead. Survival data was analyzed by Kaplan Meier method.

  Mice were subjected to unrestrained WBP (Buxco, Troy, NY). Lung function is expressed as Enhanced Pause (Penh), a unitless calculation that reflects the resistance of the lung. This value is derived from changes in the holding vessel pressure that fluctuate as a result of changes in the animal's respiratory pattern. The bronchoconstriction of the animal's airways affects the air flow and hence the pressure in the holding container. The change in pressure is tracked during exhalation (PEP) and inspiration (PIP). The Penh value is calculated according to the formula Penh = pause × PEP / PIP, where “pause” reflects the timing of expiration. Mice were acclimated for 15 minutes in a plethysmography chamber, then data were collected at 1 minute intervals, averaged over 10 minutes and expressed as absolute Penh values. To compare groups, data were analyzed using a two-way ANOVA and Bonferroni post-test. P values less than 0.05 were considered significant.

  Results: In a mouse model of influenza lung infection, compound (1) is used in combination with oseltamivir for its ability to prevent death and morbidity, reduce weight loss, and protect and / or restore lung function. 1) or oseltamivir was evaluated for single treatment. The combination did not adversely affect the efficacy of each drug compared to each drug administered alone. In addition, a failure dose for each compound alone (0.3 and 10 mg / kg of compound (1) and oseltamivir, respectively) survived from 0 percent to 100 percent when combined. As the rate increased, the combination treatment showed synergy in influenza A treatment. Compound (1) has little activity against influenza B in vivo (as expected from available in vitro data) and does not interfere with the efficacy of oseltamivir.

  Influenza A mouse model: All of the vehicle-treated controls succumbed to disease by day 9 or 10. When starting dosing at +48 hours post infection, treatment with 1, 3 and 10 mg / kg of compound (1) alone with BID provides complete protection from death and reduced weight loss compared to vehicle control And resulted in a recovery of lung function (Table 17). Treatment with 0.1 and 0.3 mg / kg of compound (1) administered alone and 10 mg / kg of oseltamivir does not protect against death when treatment is initiated +48 hours after influenza A infection or body weight Did not diminish decrease or restore lung function. Interestingly, 0.3 mg / kg Compound (1) and oseltamivir administered together +48 hours after influenza A infection provide complete protection from death, reduce weight loss and restore lung function It was.

  Influenza B mouse model: All of the vehicle-treated controls succumbed to disease by day 7 or 8. By administering 1, 3 or 10 mg / kg of compound (1) alone -2 hours prior to influenza B infection and continuously with BID for 10 days, compared to control, morbidity, weight loss or There was no significant protection from loss of lung function. Complete protection from death, reduced weight loss by administering oseltamivir alone or in combination with 1, 3 or 10 mg / kg of compound (1) at 10 mg / kg -2 hours prior to influenza B infection And resulted in recovery of lung function (Table 18).

  Example 8: In vivo assay for combined use of compound (1) and oseltamivir

Infected mice are combined with zanamivir starting 24 hours prior to influenza A challenge with 5 × 10 ³ TCID ₅₀ A / PR / 8/34 in vehicle or increasing dose levels of Form A compound (1) 1 / Treated with the HCl salt of 2H ₂ O (hereinafter simply, compound (1) in Example 8). A suspension of influenza A challenge and compound (1) was prepared in a manner similar to that described above in Example 7. Challenged mice were treated with zanamivir with 0.3 mg / kg, 1 mg / kg or 3 mg / kg IN once 24 hours prior to IN (intranasal) challenge with 5 × 10 ³ TCID ₅₀ A / PR / 8/34 And 0.1 mg / kg, 0.3 mg / kg or 1 mg / kg of compound (1 starting at -2 hours prior to challenge with 5 × 10 ³ TCID ₅₀ A / PR / 8/34 for 10 days ).

  The results are summarized in Table 19A and Table 19B below. As shown in Table 18A below, combination therapy with Compound (1) and zanamivir resulted in an additional survival effect (Table 19A). Efficiency index (% survival / (% weight loss on day 8) * (Penh on day 6)), a composite measure of survival, weight loss and lung function, is summarized in Table 19B.

  Example 9: Prophylactic efficacy and post-infection efficacy of compound (1) in mouse influenza A infection model

  Materials and methods

  Animals: Female 18-20 g BALB / c mice were obtained from Jackson Laboratories (Bar Harbor, ME) for antiviral experiments. The animals were maintained with standard free rodent chow and tap water. The animals were quarantined 48 hours before use.

  Virus: Mouse adapted influenza A / California / 04/2009 (pndH1N1) virus, Dr. Obtained from Elena Govorkova (St. Jude Children's Research Hospital, Memphis, TN). Viral stocks were amplified in MDCK cells and then titrated for lethality in BALB / c mice. Influenza A / Victoria / 3/75 (H3N2) virus was obtained from the American Type Culture Collection (Manassas, VA). The virus was inoculated 7 times in mice to match the mice and then once in MDCK cells. To obtain an appropriate lethal challenge dose, the virus was further titrated for lethality in BALB / c mice. Influenza A / Vietnam / 1203/2004 (H5N1) virus was obtained from Dr. of Centers for Disease Control (Atlanta, GA). Obtained from Jackie Katz. Mice were exposed to lethal doses of the virus (5MLD50, 5PFU / mouse). This lethal dose had previously been killed during days 6-13, and this dose had 90-100% mortality by day 10.

  Compound: Oseltamivir (as Tamiflu®) was obtained from a local pharmacy. Each capsule of Tamiflu contains 75 mg of active ingredient, ie oseltamivir carboxylate, when metabolized in the body. The dose of oseltamivir is based on this measurement. The HCl salt of Form A compound (1) hemihydrate (hereinafter simply Compound (1) in Example 9) is the subject of this study, and the dose of the compound is 1) Based on hemihydrate HCl salt. Both compound (1) and oseltamivir were prepared in 0.5% methylcellulose (Sigma, St. Louis, MO) for oral gavage (po) administration to mice.

  Experimental design: The mice were anesthetized by intraperitoneal injection of ketamine / xylazine (50/5 mg / kg) and the animals were infected with 90 μl of influenza virus suspension in the nasal cavity. Viral challenge was approximately 4 times the lethal dose of 50% mouse infection. Treatment was performed twice daily (at 12 hour intervals) for 10 days starting 2 hours prior to virus challenge or 48 hours after challenge, as indicated. Parameters for assessing infection were survival, mean date of death, weight change and lung infection parameters (bleeding score, weight and virus titer). The animals were weighed individually every other day until day 21 of infection. Mice that died during the first 6 days of the treatment period were considered to have died from causes other than influenza virus infection and were excluded from the total count.

  To assess pulmonary infection parameters, lungs of sacrificed animals (initially leaving 5 animals per group for this purpose) were collected. The pulmonary hemorrhage score was assessed by visual inspection for a color change from light red to dark purple. This occurs locally in the lung, not due to the gradual change of the entire lung to a darker color. The bleeding score ranges from 0 (normal) to 4 (the whole lung shows dark purple) and is therefore a non-parametric measurement. Lungs were weighed and then frozen at -80 ° C. The thawed lung was then homogenized in 1 ml of cell culture medium, the supernatant fluid was centrifuged to remove particulate matter, and the liquid sample was frozen again at -80 ° C. After preparing a 96-well plate of MDCK cells, samples were thawed, serially diluted in 10-fold dilution increments, and titrated by endpoint dilution method on that plate (1) using 4 microwells per dilution. . The virus titer was calculated as log 10 50% cell culture infectious dose per gram of lung tissue (log 10 CCID 50 / g).

  Statistical analysis: Statistical significance was determined by analyzing Kaplan-Meir plots for comparing multiple groups by the Mantel-Coxlogrank test. Subsequently, pairwise comparisons were made by the Gehan-Breslow-Wilcoxon test. The relative experimental significance was adjusted to the Bonferroni corrected significance threshold based on the number of treatment comparisons made. Comparison of mean date of death and mean pulmonary hemorrhage score was analyzed by Dunn's multiple comparison test followed by Kruskal-Wallis test. Mean body weight, lung weight and log 10 lung virus titer were assessed by ANOVA assuming equal variance and normal distribution. After ANOVA, individual treatment values were compared by Tukey-Kramer multiple comparison test. Analysis was performed using Prism® software (GraphPad Software, San Diego, Calif.).

  Results and Discussion

  The prophylactic dose response of compound (1) was investigated in a mouse influenza A model. Vehicle or compound (1) administration started 2 hours prior to infection and continued twice daily for 10 days. The results are summarized in Table 20 and Table 21. All mice that received vehicle alone succumbed to infection by day 9 of the study, and on average lost about 32% of body weight (BW). Compound (1) administered at 1, 3 or 10 mg / kg BID resulted in a complete survival and a dose-dependent decrease in weight loss. Compound (1) administered at 0.3 mg / kg BID produced some survival effects (2/8 mice), but those mice lost significant weight. In the same experiment, mice received oseltamivir at 10 mg / kg BID, which is a clinically equivalent human dose (based on AUC). All mice that received oseltamivir survived a similar weight loss profile as mice that received compound (1) at 1 mg / kg BID.

  Compound (1) is still effective in this model challenged with influenza A / Vietnam / 1203/2004 (H5N1) virus when administered 48 hours after infection and continued with BID for 10 days. (Table 22). Administration of Compound (1) at 10 mg / kg resulted in complete protection as shown in Table 20.

  Example 10: In vitro efficacy of compound (1) against a range of influenza strains

Cells and viruses. Madine Darby canine kidney (MDCK) cells were first obtained from the American Type Culture Collection (ATCC, Manassas, Va.) And passaged using standard laboratory techniques prior to use in infection assays. Cells were treated with Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO), 2 mM L-glutamine, 10 mM HEPES, 100 U / mL penicillin and 100 μg / mL streptomycin (Invitrogen). Maintained at 37 ° C. in Invitrogen, Carlsbad, Calif.). The influenza virus, ATCC, Centers for Disease Control and Prevention (CDC; Atlanta, the of GA Influenza Division of the Virus Surveillance and Diagnosis Branch or the Influenza Reagent Resource, Influenza Division, WHO Collaborating Center for Surveillance, Epidemiology and Control of Influenza, Obtained from CDC To make virus stocks, 2 mM L-glutamine, 10 mM HEPES, 100 U / mL penicillin, 100 μg / mL strep MDCK cells were infected with low infection efficiency (MOI) in DMEM supplemented with mycin and 1 μg / mL tolylsulfonylphenylalanyl chloromethyl ketone (TPCK) treated trypsin (USB Corp .; Santa Clara, Calif.). Was incubated at 37 ° C., 5% CO ₂ for 48 hours, after which the supernatant was collected by centrifuging at 900 × g for 10 minutes using a Beckman GS-6R centrifuge. Frozen at -80 ° C.

  Compound. Compound (1) free base or HCl salt (eg, amorphous HCl salt of compound (1), compound A (1) hemihydrate HCl salt, amorphous free base compound (1) (Hereafter, briefly, the compound (1) in Example 10) was dissolved in 100% dimethyl sulfoxide (DMSO) to prepare a 10 mM solution.

Antiviral activity. Antiviral activity of compound (1) was evaluated in MDCK cells as measured by ATP level using CellTiter-Glo (Promega; Madison, Wis.). MDCK cells were plated in black 384 well plates with clear bottoms at a density of 2 × 10 ⁴ cells per well in 50 μL VGM. Cells were allowed to adhere and form a monolayer by incubating the cells at 37 ° C., 5% CO ₂ , saturated humidity. After 5 hours, 40 μL of media was removed and 15 μL of virus was added at a MOI of 0.005. Compounds were added as 25 μL 10-point 3-fold dilutions in DMEM with supplements (final DMSO concentration of 0.5%). Internal controls consisted of wells containing only cells and wells containing untreated cells infected with virus. After 72 hours of incubation, 20 μL of CellTiter-Glo was added to each well and incubated for 10 minutes at room temperature. Luminescence was measured using an EnVision Multilabel reader (PerkinElmer; Waltham, Mass.). EC ₅₀ values (concentrations of compounds that ensure 50% cell viability of uninfected controls) were determined using a four parameter curve fitting method using the Levenburg Marquardt algorithm (Condose software; Genedata, Basel, Switzerland). Calculated by fitting to reaction data. In vitro testing of hpaiH5N1 was performed at the Southern Research Institute under BSL-3 containment.

  As shown in Table 23 below, Compound (1) is an H1N1 and H3N2 reference strain from 1934 to 2009, and H1N1 strain A / California / 07/2009, A / Texas, which was globally prevalent in 2009 Potent activity against all influenza A strains tested, including / 48/2009 and highly pathogenic avian H5N1 strain A / VN / 1203/2004. Compound (1) was equally effective against all strains, including those resistant to amantadine inhibitors and neuraminidase inhibitors. Compound (1) showed limited activity against influenza B virus.

  Example 11: In vitro combination experiment of compound (1) with oseltamivir, zanamivir or fabipyravir

  A solution of compound (1) (free base or HCl salt of compound (1) similar to that in Example 10) in 100% dimethyl sulfoxide (DMSO) was converted to neuraminidase inhibitor oseltamivir carboxylate and zanamivir or polymerase inhibition. In combination experiments with any of the agents T-705, they were tested in a 3-day MDCK cell CPE-based assay infected with A / Puerto Rico / 8/34 at a MOI of 0.01. Oseltamivir carboxylate and T-705 were dissolved in 100% dimethyl sulfoxide (DMSO); zanamivir was dissolved in Dulbecco's modified Eagle's medium (DMEM) at a concentration of 10 mM and stored at -20 ° C. In this study, Bliss independence method (Macsynergy) (eg Prichard, MN and C. Shipman, Jr., Antivirus, 199.14 (4-5): p.181-205. ) Or Loewe additivity / Median-effect method (e.g. Chou, TC and P. Talalay, Adv Enzyme Regul, 1984.22: p.27-55). Using. The Bliss-independent method involves testing combinations of different concentrations of inhibitors in a checkerboard format, and the Lowein-independent method tests a fixed ratio of inhibitor combinations at various dilutions of that fixed ratio. Including that. Experiments using a combination of compound (1) and itself as a control were also performed to confirm the additivity. Cell viability was measured using CellTiter-Glo.

  The Bliss-independent method resulted in a magnitude of synergy of 312 and 268 for oseltamivir carboxylate and zanamivir respectively; a magnitude of 317 for favipiravir was obtained. A magnitude of synergy greater than 100 is usually considered strong synergy and a magnitude of 50-100 is considered moderate synergy. The Loewe additive method has a C.C. of 0.58, 0.64 and 0.89 for oseltamivir, zanamivir and T-705, respectively, at 50% effective level. I. (Combined index) value. C. less than 0.8 I. Values are considered strong synergies, and values between 0.8 and 1.0 are considered additive to somewhat synergistic. Together these data suggest that compound (1) is synergistic with the neuraminidase and polymerase inhibitors tested, as shown in Table 24.

  Example 12: Efficacy in mouse influenza A infection model

  The prophylactic dose response of compound (1) (amorphous or A form of compound (1) hemihydrate HCl salt (hereinafter concisely compound (1) in the examples) was In the model, vehicle or compound (1) administration was started 2 hours prior to infection and continued twice daily for 10 days All mice receiving vehicle alone were treated on day 9 of the study. By average, about 32% of body weight (BW) was reduced, compound (1) administered at 1, 3 or 10 mg / kg BID was dose dependent for complete survival and weight loss Compound (1) administered at 0.3 mg / kg BID produced some survival effect (2/8 mice), but those mice lost significant weight. In the same experiment, Mice received oseltamivir at 10 mg / kg BID, which is a clinically equivalent human dose (based on AUC) All mice that received oseltamivir received compound (1) at 1 mg / kg BID Survived with a weight loss profile similar to mice.

  In this model, mice are challenged with influenza A virus to the extent that administration of compound (1) can be delayed and still provide efficacy, and administration of vehicle, oseltamivir or compound (1) is 24 The study was conducted by continuing BID administration for 10 days starting 48, 72, 96 or 120 hours (Table 25). All vehicle controls succumbed to disease by day 8 or 9 of the study. When dosing is started by 72 hours after infection, administration of compound (1) at 1, 3 or 10 mg / kg BID provides complete protection from death and reduced weight loss compared to vehicle control. It was brought. When dosing was started 24 hours after infection or earlier, administration of oseltamivir at 10 mg / kg BID alone provided complete protection. Compound (1) at 3 or 10 mg / kg BID resulted in complete survival 96 hours after infection when further delaying the onset of compound administration and partial when start of dosing was delayed 120 hours after infection. Brought protection.

  The effectiveness of the compound (1) for reducing the viral titer of the lung was examined. Mice were infected with influenza A and 24 hours later, vehicle, oseltamivir (10 mg / kg BID) or compound (1) (3, 10, 30 mg / kg BID) were measured for lung recovery and viral load on day 6. (Table 26). All groups receiving Compound (1) showed a robust statistically significant reduction in lung viral titers compared to animals receiving oseltamivir and vehicle.

To establish the PK / PD model, mice were infected with influenza virus for 24 hours and then compound (1) was administered for an additional 24 hours. Doses were single or divided into 2 or 4 doses administered every 12 or 6 hours, respectively. Lungs and plasma were collected to determine lung viral load and compound (1) concentration. Individual lung titer data from these dosing regimens (q6h, q12h and q24h) were plotted against individual C _max C _min or AUC values (data not shown). There was a clear correlation between the decrease in lung titer and _Cmin , but there was little correlation with _Cmax and only a weak correlation with AUC. When the measured concentration of compound (1) in plasma was plotted against the measured lung titer, there was a strong correlation with _Cmin . A half-maximal decrease in lung titer (2-3 log) occurs around the serum-shifted EC ₉₉ (100 ng / mL). A similar correlation was found between lung titer and compound (1) concentration measured in the lung (data not shown).

  Example 13: Proof of concept for influenza challenge

  A live attenuated influenza challenge model was previously used to predict the efficacy of influenza antiviral agents in natural human infections (Calfee, DP, Peng, AW, Hussey, E K., Lobo, M. & Hayden F. G. Safety and effectiveness of once daily in vivo random in preventing expr. 99. H. Den. Use of the oral neurominidase inhibitor osaltamivir in experimental human influenza.JAMA 282, 1240-1246 (1999) Form A compound in healthy volunteers inoculated with live influenza A / Wisconsin / 67/2005 (H3N2) challenge strain virus (1) Hemihydrate HCl A randomized, double-blind, placebo-controlled, single-center study of salt (hereinafter simply Compound (1) in this example in this example) was conducted and subjects received a daily dose of placebo (N = 33). Or any one of compound (1) administered once a day (QD) 5 times (in the form of a capsule consisting of undiluted compound (1)): 100 mg (N = 16), 400 mg on day 1 (N = 19) or 900 mg, followed by 600 mg on days 2-5 (N = 20), or 1200 mg on day 1, followed by 600 on days 2-5 mg (N = 18) Subjects received nasal swabs three times daily, scored for clinical symptoms three times daily on days 1-7 and released from the facility on day 8, approximately 28 Safety follow-up was performed on day 1. Nasal swabs were assayed for influenza virus by cell culture (primary analysis) and qRT-PCR (secondary analysis).

Efficacy analysis is that at least one dose of the test drug (compound (1) or placebo) was received and the virus concentration was above the lower limit of quantitation for the TCID ₅₀ cell culture assay at any time within 48 hours after inoculation. The largest analyst, defined as all randomized subjects who were or were equal, or whose hemagglutination inhibition titers were increased 4 fold or more from baseline (day 1) in the post-inoculation period (FA) was performed on the population (N = 74). The safety set included all subjects who were vaccinated with influenza on day 0 and received at least one dose of either placebo or compound (1) (N = 104). .

  Evaluation of effectiveness

The primary measure in this study is the trend in dose response in the AUC of viral shedding from day 1 (first day of drug administration) to day 7 of the study as measured by TCID ₅₀ in the cell culture assay in the FA population. It was a demonstration. A statistically significant dose response trend was observed in the median AUC virus shedding in nasal swabs (P = 0.036, Jonkheel Tapstra trend test). In addition, pairwise comparisons were made between the combined placebo group and each dose group of Compound (1) for the median AUC virus shedding, median shedding duration, and average magnitude of virus shedding peak (Table 27). ). A statistically significant decrease in AUC virus shedding was observed for the 1200/600 mg dose group (P = 0.010, Wilcoxon rank sum test), and a significant decrease in peak shedding was seen in the 1200/600 mg dose group ( FIG. 13) was observed for the 400 mg dose group and the combined compound (1) dose group. A further FA group analysis was performed (data not shown).

  Nasal influenza excretion was also quantified by qRT-PCR and the results were similar to those observed in cell culture. There is no difference in the rate of seroconversion, defined by a 4-fold increase or more in the anti-influenza titer from baseline prior to inoculation, between the compound (1) dose group and the placebo. It is suggested that Compound (1) administered after time did not affect the rate of acquisition of influenza infection and did not abolish the humoral immune response to subsequent infection (Table 28).

  Subjects recorded clinical symptoms in a diary three times a day. The AUC of the clinical score and the influenza-like symptom score on the first day to the seventh day was calculated. Compared to placebo, the 1200/600 mg dose group of compound (1) has a median duration of complex clinical symptoms (P = 0.001), median AUC of influenza-like symptoms (P = 0.040) And a statistically significant decrease in median duration of influenza-like symptoms (P <0.001) (Table 28).

ＡＵＣ：値−時間曲線下面積（ａｒｅａ ｕｎｄｅｒ ｔｈｅ ｖａｌｕｅ ｖｅｒｓｕｓ ｔｉｍｅ ｃｕｒｖｅ）；ＣＩ：信頼区間；ＮＡ：該当なし；ｑＲＴ−ＰＣＲ：定量的逆転写酵素ポリメラーゼ連鎖反応；ＳＤ：標準偏差；ＴＣＩＤ５０：５０％組織培養感染量
注：統計学的に有意なＰ値（Ｐ＜０．０５）は太字である。
^ａヨンクヒール・タプストラ傾向検定からのＡＵＣの用量反応傾向についてＰ＝０．０３６。
^ｂウィルコクソン順位和検定から算出されたＰ値。
^ｃＡＮＯＶＡから算出されたＰ値。
^ｄログランク検定から算出されたＰ値。
^ｅヨンキー（Ｊｏｎｃｋｈｅｒｒｅ）・テルプストラ傾向性検定からのＡＵＣの用量反応傾向についてＰ＝０．０３１。
^ｆセロコンバージョンは、経過観察の来院時の抗インフルエンザ抗体価の、ベースラインと比較したときの≧４倍増加として定義される。フィッシャーの正確確率検定を用いて算出されたＰ値。

AUC: area under the value versus time curve; CI: confidence interval; NA: not applicable; qRT-PCR: quantitative reverse transcriptase polymerase chain reaction; SD: standard deviation; TCID50: 50% Tissue culture infectious dose Note: Statistically significant P values (P <0.05) are in bold.
^a P = 0.036 for AUC dose-response trend from the Yonkhheel Tapsta trend test.
^b P value calculated from Wilcoxon rank sum test.
^c P value calculated from ANOVA.
^d P value calculated from log rank test.
^e P = 0.031 for AUC dose response trend from Joncherre terpstra propensity test.
^f Seroconversion is defined as a ≧ 4 fold increase in anti-influenza antibody titer at the follow-up visit compared to baseline. P value calculated using Fisher's exact test.

ＡＵＣ：値−時間曲線下面積；ＣＩ：信頼区間；ＮＡ：該当なし。
注：統計学的に有意なＰ値（Ｐ＜０．０５）は太字である。
^ｂウィルコクソン順位和検定から算出されたＰ値。
^ｃＡＮＯＶＡから算出されたＰ値。
^ｄログランク検定から算出されたＰ値。

AUC: area under the value-time curve; CI: confidence interval; NA: not applicable.
Note: Statistically significant P values (P <0.05) are in bold.
^b P value calculated from Wilcoxon rank sum test.
^c P value calculated from ANOVA.
^d P value calculated from log rank test.

  Safety assessment

  Compound (1) was well tolerated, there was no interruption due to adverse events (AE) associated with compound (1), and there were no serious adverse events. A list of adverse events occurring in ≧ 10% of subjects in any treatment group is presented (Table 29). Influenza-like illness was the most frequently reported adverse event and was reported by approximately equal proportion of subjects in the placebo and compound (1) groups. Adverse events that occurred with a ≥10% incidence difference between the Compound (1) group and placebo recipients resulted in decreased blood phosphorus levels (18.1%, Compound (1); 0%, placebo) Nasal discharge (compound (1), 4.2%; 18.8%, placebo) and nasal congestion (1.4%, compound (1); 15.6% placebo). In addition, an increase in alanine aminotransferase (ALT) was observed in both placebo and compound (1) recipients. No abnormal liver function or decreased serum phosphate was observed in a first in human dose escalation study of Compound (1) in daily single doses up to 1600 mg and multiple doses up to 800 mg daily for 10 days; Both an increase in ALT and a decrease in serum phosphate have been previously reported in upper respiratory tract virus infection.

注：複数の事象を有する被験体は、ＡＥのもとで一度カウントされた。被験体は、複数のカテゴリーに見られることがある。
^ａ１日目における９００ｍｇの単回の負荷量および２〜５日目におけるｑｄの６００ｍｇ。
^ｂ１日目における１２００ｍｇの単回の負荷量および２〜５日目におけるｑｄの６００ｍｇ。
^ｃ有効性の解析において定義されたようなインフルエンザ様疾病を本文に列挙されたパラメータに基づいて評価した。インフルエンザ様疾病のＡＥは、医師によって判定された。

Note: Subjects with multiple events were counted once under AE. Subjects may be found in multiple categories.
^a Single load of 900 mg on day 1 and 600 mg of qd on days 2-5.
^b Single loading of 1200 mg on day 1 and 600 mg of qd on days 2-5.
^c Influenza-like illnesses as defined in the efficacy analysis were evaluated based on the parameters listed in the text. The AE for influenza-like illness was determined by a physician.

  Consideration

In an influenza challenge study in healthy volunteers, compound (1) showed a dose response trend of AUC virus titer in nasal swabs by both TCID ₅₀ cell culture and qRT-PCR, with the highest dose of compound (1 ) Caused a significant reduction in AUC virus titer and AUC and duration of influenza symptoms. A similar magnitude improvement over placebo was not observed in the second highest dose group, 900/600 mg (Table 27), but this dose was not a central AUC for combined clinical and influenza-like symptoms endpoints. Regarding values, results were similar to the 1200/600 mg dose (Table 28); the reason for this discrepancy is not fully understood. Although no positive safety trend was encountered in the POC study, the observed decrease in phosphate and increase in ALT suggest that proper monitoring of both parameters is necessary for use in future studies. To do.

  Overall, the limitation of the influenza challenge model is that the influenza virus used in this study is a strain specifically selected so that it does not result in the most severe clinical symptoms of influenza virus infection . Further, the administered viral inoculum is probably more than the inoculum in natural influenza exposure. In social environments where patients often do not seek diagnosis or treatment until they develop substantial symptoms (which is probably more than 24 hours after exposure), the timing of administering Compound (1) 24 hours after exposure is It may not be a realistic time frame for the start of treatment. However, time scales cannot be directly compared given that naturally infected subjects are initially inoculated with a much lower virus titer.

  In summary, compound (1) is a potent influenza A PB2 inhibitor that represents a unique new class of antiviral agents. The properties of this inhibitor explained by both preclinical and clinical data indicate that compound (1) has several potential advantages over current antiviral agents used to treat influenza infections. To be an interesting candidate for further evaluation.

  All references provided herein are hereby incorporated by reference in their entirety. As used herein, all abbreviations, symbols and conventions are consistent with those used in modern scientific literature. For example, Janet S. et al. Dodd, ed. The ACS Style Guide: A Manual for Authors and Editors, 2nd Ed. , Washington, D .; C. : See American Chemical Society, 1997.

  Example 14: Compound (1) enriched in deuterium

  Compound (1) enriched in deuterium was synthesized according to Scheme 1 below:

Scheme 1:

Reagents and conditions: (Step 14a) Maleic anhydride, CHCl ₃ ; (Step 14b) Beta-quinine, ethanol, toluene; (Step 14c) DPPA, Et ₃ N, 90 ° C., BnOH; (Step 14d) H ₂ , Pd (Step 14e) amine 14-5, ⁱ Pr ₂ NEt, THF, 70 ° C; (Step 14f) HCl, dioxane, MeCN, 80 ° C; (Step 14g) NaOH, THF, MeOH.

14A: Compound (14-1)

Potassium tert-butoxide (9.663 g, 86.11 mmol) was dissolved in DMSO-d ₆ (30.00 mL) and placed under nitrogen. A solution of cyclohexa-1,4-diene (6 g, 74.88 mmol) in pentane (60.00 mL) was added and the mixture was stirred under nitrogen for 2.5 hours. The DMSO-d ₆ layer was removed and a new 30 mL DMSO-d ₆ containing potassium tert-butoxide (9.663 g, 86.11 mmol) was added. Stirring was continued overnight. The layers are separated and the pentane layer is washed with D ₂ O (50 mL) and dried over Na ₂ SO ₄ to give 1,2,3,4,5,5,6,6-octauteuteriocyclohexa- 1,3-diene (14-1) was obtained and proceeded to the next step as a solution. This reaction forms a mixture of the 1,3-diene isomer and the 1,4-diene isomer. Only 1,3-diene reacts in subsequent steps.

14b: 3a, 4,7,7a-tetrahydro-4,7-ethanoisobenzofuran-1,3-dione-4,5,6,7,8,8,9,9-d ₈ (14-2)

A pentane solution of 1,2,3,4,5,5,6,6-octadeuteriocyclohexa-1,3-diene (14-1) (6.5 g, 74.0 mmol) in chloroform (50 mL). Diluted and treated with maleic anhydride (8.0 g, 81.4 mmol). The reaction mixture was stirred at room temperature overnight. The solvent was evaporated under reduced pressure and the resulting semi-solid residue was treated with MeOH. After stirring for 10 minutes, the MeOH slurry was cooled to approximately 20 ° C. The resulting precipitate was collected by filtration and washed three times with a small amount (5 mL) of cold methanol to give the product (14-2) as a white solid: ¹ H NMR analysis (CDCl ₃ ) 3. 15 (s, 2H) indicates a clean product and 95% deuterium incorporation.

14c: (+/−)-trans-3- (ethoxycarbonyl) bicyclo [2.2.2] oct-5-ene-2-carbon-1,4,5,6,7,7,8,8- d ₈ acid (14-3)

A dropping funnel and an internal temperature probe were attached to a 3-neck RBF under nitrogen. To the flask, 3a, 4,7,7a-tetrahydro-4,7-ethanoisobenzofuran-1,3-dione-4,5,6,7,8,8,9,9-d ₈ (14-2 ) (2.68 g, 14.39 mmol), beta-quinine (5.24 g, 15.83 mmol) and anhydrous toluene (40 mL). The reaction was stirred magnetically and cooled to −25 ° C. (cold finger cooling). A solution of absolute ethanol (8.40 mL, 143.90 mmol) in anhydrous toluene (13.4 mL) was added over 25 minutes while maintaining the internal temperature below -25 ° C. The reaction mixture was stirred at approximately −20 ° C. overnight. The precipitated gel-like solid was collected by filtration, washed with toluene (3 × 30 mL), and then dissolved in 1N aqueous HCl / EtOAc (300 mL of 1: 1 mixture). The biphasic mixture was stirred until all the precipitate had dissolved. The layers were separated and the organic layer was washed with water (2 × 100 mL), brine (100 mL), dried over Na ₂ SO ₄ , filtered and concentrated at low temperature on a rotavapor to give 800 mg of desired Product (14-3) was obtained and used without further purification.

14d: (+/−)-trans-ethyl-3-(((benzyloxy) carbonyl) amino) bicyclo [2.2.2] oct-5-en-2-carboxylate-1,4,5,6 , 7, 7, _{8, 8} -d ₈ (14-4)

(+/−)-trans-3- (ethoxycarbonyl) bicyclo [2.2.2] oct-5-ene-2-carboxylic-1,4,5,6,7, in toluene (4.5 mL) _{7,8,8-d 8} acid, (14-3) (0.60g, 2.58mmol ) in a solution of diphenylphosphoryl azide (0.81 g, 0.63 mL, 2.84 mmol) was added, triethylamine (0.40 mL, 2.84 mmol) was added. The reaction mixture was heated to 90 ° C. for 2 hours. To the mixture was added benzyl alcohol (0.35 mL, 3.34 mmol) and it was heated at 90 ° C. overnight. The reaction mixture was cooled to room temperature and partitioned between EtOAc and saturated aqueous NaHCO ₃ solution. The layers were separated and the organic phase was washed with saturated aqueous NH ₄ Cl, brine, dried over Na ₂ SO ₄ , filtered and evaporated to dryness. The crude residue was purified by silica gel chromatography (0-35-100% EtOAc / hexanes-stained with CAMA). ¹ H NMR shows the desired product (14-4) with the benzyl alcohol impurity still present. The material was advanced without further purification.

14e: (+/−)-trans-ethyl-3-aminobicyclo [2.2.2] octane-2-carboxylate-1,4,5,5,6,6,7,8-d ₈ (14 -5)

Palladium (0.052 g, 0.049 mmol) was charged into a hydrogenation vessel (under nitrogen atmosphere) and wetted with approximately 5 mL of methanol. To the suspension was added (+/−)-trans-ethyl (2S, 3S) -3-(((benzyloxy) carbonyl) -amino) bicyclo [2.2.2] octa- in methanol (20 mL). A solution of 5-ene-2-carboxylate-1,4,5,6,7,7,8,8-d ₈ (14-4) (0.521 g, 1.547 mmol) was added. The reaction mixture was subjected to hydrogenation (44 PSI) overnight. The pressure was released and the catalyst was filtered off. All volatiles were removed in vacuo. The crude product (14-5) was used without further purification.

14f: (+/−)-trans-ethyl-3-((5-fluoro-2- (5-fluoro-1-tosyl-1H-pyrrolo [2,3-b] pyridin-3-yl) pyrimidine-4 - yl) amino) bicyclo [2.2.2] octane-2-carboxylate _{-1,4,5,5,6,6,7,8-d} 8 (14-7)

(+/-)-trans-ethyl (2S, 3S) -3-aminobicyclo [2.2.2] octane-2-carboxylate-1,4,5,5,6,6 in THF (10 mL) , 7,8-d ₈ (14-5) (0.317 g, 1.547 mmol) and 5-fluoro-3- (5-fluoro-4-methylsulfinyl-pyrimidin-2-yl) -1- (p- To a suspension of (tolylsulfonyl) pyrrolo [2,3-b] pyridine (14-6) (0.694 g, 1.547 mmol) was added N, N-diisopropylethylamine (0.808 mL, 4.641 mmol), The reaction mixture was heated to 70 ° C. overnight. The reaction mixture was diluted with EtOAc and water. The layers were separated and the organic phase was washed with brine, dried (MgSO ₄ ), filtered and concentrated in vacuo. The crude product (14-7) was purified by silica gel chromatography (0-100% EtOAc / hexanes) to give the desired product.

14 g: (+/−)-trans-ethyl-3-((5-fluoro-2- (5-fluoro-1H-pyrrolo [2,3-b] pyridin-3-yl) pyrimidin-4-yl) amino ) bicyclo [2.2.2] octane-2-carboxylate _{-1,4,5,5,6,6,7,8-d} 8 (14-8)

(+/-)-trans-ethyl (2S, 3S) -3-((5-fluoro-2- (5-fluoro-1-tosyl-1H-pyrrolo [2,3-b] in acetonitrile (6 mL)) pyridin-3-yl) pyrimidin-4-yl) amino) bicyclo [2.2.2] octane-2-carboxylate _{-1,4,5,5,6,6,7,8-d} 8 (14- 7) To a solution of (373 mg, 0.6325 mmol) was added HCl (800 μL of a 4 M solution in dioxane, 3.200 mmol). The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was then heated to 80 ° C. for 6 hours, then cooled to room temperature and stirred overnight. LC / MS analysis indicated that the reaction was incomplete. To the mixture was added an additional 6 ml CH ₃ CN and 800 μl 4N HCl / dioxane solution. The reaction mixture was heated to 80 ° C. for 4 hours. All volatiles were removed under reduced pressure and the residue was diluted with EtOAc and saturated aqueous NaHCO ₃ . The layers were separated and the organic phase was washed with brine, dried over MgSO ₄ , filtered and concentrated in vacuo. The crude residue was purified by silica gel chromatography (0-100% EtOAc / hexanes) to give the desired product (14-8):

¹ H NMR (300 MHz, d6-DMSO) δ 12.28 (s, 1H), 8.50 (dd, J = 9.8, 2.8 Hz, 1H), 8.23 (ddd, J = 12 .6, 6.2, 2.7 Hz, 2H), 7.60 (d, J = 6.9 Hz, 1H), 4.73 (t, J = 6.5 Hz, 1H), 4.30. -3.85 (m, 2H), 2.89 (d, J = 6.8 Hz, 1H), 1.59-0.96 (m, 4H).

14h: (+/−)-trans-ethyl-3-((5-fluoro-2- (5-fluoro-1H-pyrrolo [2,3-b] pyridin-3-yl) pyrimidin-4-yl) amino ) bicyclo [2.2.2] octane-2-carboxylic _{-1,4,5,5,6,6,7,8-d 8} acid (1)

(+/−)-trans-ethyl-3-((5-fluoro-2- (5-fluoro-1H-pyrrolo [2,3-b]) dissolved in THF (3.0 mL) and methanol (1 mL) pyridin-3-yl) pyrimidin-4-yl) amino) bicyclo [2.2.2] octane-2-carboxylate _{-1,4,5,5,6,6,7,8-d} 8 (14- 8) To a solution of (0.165 g, 0.379 mmol) was added NaOH (1 mL of 2M solution, 2.000 mmol) and the reaction mixture was stirred at room temperature for 3 h. LC / MS analysis indicates that the reaction is incomplete. The reaction mixture was warmed to 45 ° C. for 2 hours and then warmed to 55 ° C. for 30 minutes. The reaction mixture was diluted in saturated aqueous NH ₄ Cl. The pH was adjusted to approximately 6.5 by adding a few drops of 1N HCl. The product was extracted with EtOAc. The organic phase was dried over MgSO ₄ , filtered and concentrated in vacuo to give the desired product (1) (97.5% purity by NMR, LC / MS and HPLC): ¹ H NMR (300 MHz, d6-DMSO) δ 12.30 (d, J = 14.2 Hz, 2H), 8.79-7.94 (m, 4H), 7.58 (s, 1H), 4.68 (S, 1H), 2.84 (s, 1H), 1.85 (d, J = 85.0 Hz, 1H), 1.58-1.05 (m, 2H).

  Example 15: Compound (1) enriched in deuterium

  Alternatively, compound (1) enriched in deuterium can be synthesized according to Scheme 2 below:

Scheme 2:

Other Embodiments While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate the invention and is intended to limit the scope of the invention. Rather, it should be understood that the scope of the invention is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims (82)

Polymorphic compound (1) or a pharmaceutically acceptable salt thereof, wherein compound (1) has the following structural formula:

And the polymorph is
Compound A in Form A (1) .1 / 2H ₂ O HCl salt,
Form F compound (1). HCl salt of 3H ₂ O,
HCl salt of compound (1) in form D,
A polymorphic compound (1) or a pharmaceutically acceptable salt thereof, selected from the group consisting of Form A compound (1) and a tosylate salt of Form A compound (1). The polymorph of claim 1, wherein the polymorph is the HCl salt of Form A compound (1) .1 / 2H ₂ O. The HCl salt of Form A compound (1) .1 / 2H ₂ O is 10.5 ± 0.2, 5.2 ± 0.2, 7.4 ± 0.2 and 18 in the powder X-ray diffraction pattern. The polymorph of claim 2 characterized by one or more peaks corresponding to 2-theta values measured in units of .9 ± 0.2 degrees. The HCl salt of Form A compound (1) .1 / 2H ₂ O is 25.2 ± 0.2, 16.5 ± 0.2, 18.1 ± 0.2 and 23 in the powder X-ray diffraction pattern. 4. The polymorph of claim 3, further characterized by one or more peaks corresponding to 2-theta values measured in units of 0.0 ± 0.2 degrees. The HCl salt of Form A compound (1) .1 / 2H ₂ O is 29.2 ± 0.3 ppm, 107.0 ± 0.3 ppm, 114.0 ± 0.3 ppm and 150.5 ppm in the C ¹³ SSNMR spectrum. 5. A polymorph according to any of claims 2 to 4, characterized by one or more peaks corresponding to 7 ± 0.3 ppm. The HCl salt of Form A compound (1) .1 / 2H ₂ O has 22.1 ± 0.3 ppm, 24.6 ± 0.3 ppm, 47.7 ± 0.3 ppm and 54. ± 0.3 ppm in the C ¹³ SSNMR spectrum. 6. The polymorph of claim 5, further characterized by one or more peaks corresponding to 8 ± 0.3 ppm. The polymorph according to claim 1, wherein the polymorph is an HCl salt of compound (1) · 3H ₂ O in Form F. The HCl salt of Form F compound (1) .3H ₂ O has 7.1 ± 0.2, 11.9 ± 0.2, 19.2 ± 0.2 and 12.4 in the powder X-ray diffraction pattern. 8. A polymorph according to claim 7, characterized by one or more peaks corresponding to 2-theta values measured in units of ± 0.2 degrees. Form F compound (1) 3H ₂ O HCl salt in powder X-ray diffraction pattern in units of 16.4 ± 0.2, 21.8 ± 0.2 and 23.9 ± 0.2 degrees 9. The polymorph of claim 8, further characterized by one or more peaks corresponding to the measured 2-theta values. The HCl salt of Form F compound (1) .3H ₂ O is 20.7 ± 0.3 ppm, 27.4 ± 0.3 ppm, 104.8 ± 0.3 ppm, 142.5 ± in the C ¹³ SSNMR spectrum. 10. A polymorph according to any one of claims 7 to 9, characterized by one or more peaks corresponding to 0.3 ppm and 178.6 ± 0.3 ppm. Form F compound (1). HCl salt of 3H ₂ O was found to be 154.3 ± 0.3 ppm, 20.3 ± 0.3 ppm, 132.3 ± 0.3 ppm and 21.1 ± in the C ¹³ SSNMR spectrum. 11. A polymorph according to claim 10, further characterized by one or more peaks corresponding to 0.3 ppm. The polymorph of claim 1, wherein the polymorph is the HCl salt of Compound D (1). The HCl salt of Form D compound (1) was measured in the powder X-ray diffraction pattern in units of 5.8 ± 0.2, 19.5 ± 0.2 and 17.1 ± 0.2 degrees 2 13. A polymorph according to claim 12, characterized by one or more peaks corresponding to theta values. The HCl salt of Form D compound (1) was measured in units of 5.3 ± 0.2, 10.5 ± 0.2 and 15.9 ± 0.2 degrees in the powder X-ray diffraction pattern 2 The polymorph of claim 13 further characterized by one or more peaks corresponding to theta values. The HCl salt of Form D compound (1) is 29.4 ± 0.3 ppm, 53.4 ± 0.3 ppm, 113.3 ± 0.3 ppm, 135.4 ± 0.3 ppm and in the C ¹³ SSNMR spectrum. 15. A polymorph according to any of claims 12 to 14, characterized by one or more peaks corresponding to 177.8 ± 0.3 ppm. The HCl salt of compound (1) in Form D was found to be 22.9 ± 0.3 ppm, 23.9 ± 0.3 ppm, 26.0 ± 0.3 ppm and 31.6 ± 0.3 ppm in the C ¹³ SSNMR spectrum. 16. The polymorph of claim 15, further characterized by a corresponding one or more peaks. The polymorph of claim 1, wherein the polymorph is Compound A (1). The 2-theta value of Compound A (1) measured in units of 15.5 ± 0.2, 18.9 ± 0.2 and 22.0 ± 0.2 degrees in the powder X-ray diffraction pattern 18. A polymorph according to claim 17, characterized by one or more peaks corresponding to. Form A compound (1) has units of 11.8 ± 0.2, 16.9 ± 0.2, 25.5 ± 0.2 and 9.1 ± 0.2 degrees in the powder X-ray diffraction pattern 19. The polymorph of claim 18, further characterized by one or more peaks corresponding to 2-theta values measured in In the C ¹³ SSNMR spectrum, Form A compound (1) is 21.0 ± 0.3 ppm, 28.5 ± 0.3 ppm, 50.4 ± 0.3 ppm, 120.8 ± 0.3 ppm, 138.5 20. A polymorph according to any of claims 17 to 19, characterized by one or more peaks corresponding to ± 0.3 ppm and 176.2 ± 0.3 ppm. Form A compound (1) corresponds to 30.1 ± 0.3 ppm, 25.9 ± 0.3 ppm, 22.8 ± 0.3 ppm and 25.0 ± 0.3 ppm in the C ¹³ SSNMR spectrum. 21. The polymorph of claim 20, further characterized by one or more peaks. The polymorph of claim 1, wherein the polymorph is a tosylate salt of Form A compound (1). The tosylate salt of Form A compound (1) shows 7.2 ± 0.2, 9.3 ± 0.2, 13.7 ± 0.2, 14.3 ± 0.2 in the powder X-ray diffraction pattern. 2-theta measured in units of 14.7 ± 0.2, 16.9 ± 0.2, 18.7 ± 0.2, 26.3 ± 0.2 and 26.9 ± 0.2 degrees. 23. A polymorph according to claim 22, characterized by one or more peaks corresponding to the value. The tosylate salt of Form A compound (1) was measured in units of 6.0 ± 0.2, 28.0 ± 0.2 and 27.5 ± 0.2 degrees in the powder X-ray diffraction pattern 2 The polymorph of claim 23, further characterized by one or more peaks corresponding to theta values. A pharmaceutical composition comprising the polymorph of claim 1 and at least one pharmaceutically acceptable carrier or excipient. The polymorph, A form of Compound (1) is a · 1 / 2H ₂ O of HCl salt, pharmaceutical composition according to claim 25. The polymorph, F form of Compound (1) is a · 3H ₂ O in HCl salt, pharmaceutical composition according to claim 25. 26. The pharmaceutical composition according to claim 25, wherein the polymorph is the HCl salt of compound (1) in form D. 26. The pharmaceutical composition according to claim 25, wherein the polymorph is Compound A (1). 26. The pharmaceutical composition of claim 25, wherein the polymorph is a tosylate salt of Form A compound (1). 25. A method of reducing the amount of influenza virus in a biological in vitro sample or subject, said method comprising an effective amount in said sample of a polymorphic compound (1) according to any one of claims 1-24. ). 25. A method of inhibiting influenza virus replication in a biological in vitro sample or subject, said method comprising an effective amount of said polymorphic compound (1) in said sample. ). 25. A method of treating influenza in a subject comprising administering to the subject a therapeutically effective amount of the polymorphic compound (1) of any one of claims 1-24. ,Method. 34. The method of any one of claims 31-33, further comprising co-administering one or more additional therapeutic agents to the subject. 35. The method of claim 34, wherein the additional therapeutic agent comprises an antiviral agent. 36. The method of claim 35, wherein the antiviral agent is a neuraminidase inhibitor. 37. The method of claim 36, wherein the neuraminidase inhibitor is oseltamivir or zanamivir. 36. The method of claim 35, wherein the antiviral agent is a polymerase inhibitor. 40. The method of claim 38, wherein the polymerase inhibitor is favipiravir. 40. The method according to any one of claims 31 to 39, wherein the influenza virus is an influenza A virus. A method for preparing HCl salt of Form A compound (1) .1 / 2H ₂ O, wherein compound (1) has the following structural formula:

Wherein the method comprises mixing HCl with compound (1) in a solvent system comprising water and one or more organic solvents, wherein the solvent system comprises 0.05-0. A method having a water activity of .85. The solvent system is chlorobenzene, cyclohexane, 1,2-dichloroethene, dichloromethane, 1,2-dimethoxyethane, N, N-dimethylacetamide, N, N-dimethylformamide, 1,4-dioxane, 2-ethoxyethanol, Formamide, hexane, 2-methoxyethanol, methylbutylketone, methylcyclohexane, N-methylpyrrolidone, nitromethane, pyridine, sulfolane, tetrahydrofuran (THF), tetralin, toluene, 1,1,2-trichloroethene and xylene, acetic acid, acetone Anisole, 1-butanol, 2-butanol, butyl acetate, tert-butyl methyl ether, cumene, heptane, isobutyl acetate, isopropyl acetate, methyl acetate, 3-methyl-1-butanol, methyl ethyl Selected from ketone, methyl isobutyl ketone, 2-methyl-1-propanol, ethyl acetate, ethyl ether, ethyl formate, pentane, 1-pentanol, 1-propanol, 2-propanol, propyl acetate or any combination thereof 42. The method of claim 41, comprising one or more organic solvents. The solvent system is chlorobenzene, cyclohexane, 1,2-dichloroethane, dichloromethane, 1,2-dimethoxyethane, formamide, hexane, 2-methoxyethanol, methylbutylketone, methylcyclohexane, nitromethane, tetralin, xylene, toluene, 1, 1,2-trichloroethane, acetone, anisole, 1-butanol, 2-butanol, butyl acetate, tert-butyl methyl ether, cumene, ethanol, ethyl acetate, ethyl ether, ethyl formate, heptane, isobutyl acetate, isopropyl acetate, methyl acetate , 3-methyl-1-butanol, methyl ethyl ketone, 2-methyl-1-propanol, pentane, 1-propanol, 1-pentanol, 2-propanol, propyl acetate, tetrahydrofuran Containing one or more organic solvents selected from methyl tetrahydrofuran, or any combination thereof, The method of claim 42. The solvent system is 2-ethoxyethanol, ethylene glycol, methanol, 2-methoxyethanol, 1-butanol, 2-butanol, 3-methyl-1-butanol, 2-methyl-1-propanol, ethanol, 1-pentanol, From 1-propanol, 2-propanol, methyl butyl ketone, acetone, methyl ethyl ketone, methyl isobutyl ketone, butyl acetate, isobutyl acetate, isopropyl acetate, methyl acetate, ethyl acetate, propyl acetate, pyridine, toluene, xylene or any combination thereof 43. The method of claim 42, comprising one or more selected organic solvents. 43. The method of claim 42, wherein the solvent system comprises one or more organic solvents selected from acetone, n-propanol, isopropanol, isobutyl acetate, acetic acid, or any combination thereof. 43. The method of claim 42, wherein the solvent system comprises one or more organic solvents selected from acetone or isopropanol. 47. A method according to any one of claims 41 to 46, wherein the solvent system has a water activity value of 0.4 to 0.6. 48. A method according to any one of claims 41 to 47, wherein the mixing is performed at a temperature in the range of 5C to 75C. 49. The method according to any one of claims 41 to 48, wherein the HCl is input as an aqueous solution having 30 wt% to 40 wt% HCl based on the weight of the aqueous solution. A method for preparing an HCl salt of Form F Compound (1) .3H ₂ O, wherein Compound 1 has the following structural formula:

The method is represented by
(A) mixing HCl and compound (1) in a solvent system comprising water, wherein the solvent system has a water activity equal to or greater than 0.9; or (B) stirring the HCl salt of Form A compound (1) · 1 / 2H ₂ O in a solvent system comprising water, wherein the solvent system is equal to or greater than 0.9 Having a water activity greater than The solvent system is chlorobenzene, cyclohexane, 1,2-dichloroethene, dichloromethane, 1,2-dimethoxyethane, N, N-dimethylacetamide, N, N-dimethylformamide, 1,4-dioxane, 2-ethoxyethanol, Formamide, hexane, 2-methoxyethanol, methylbutylketone, methylcyclohexane, N-methylpyrrolidone, nitromethane, pyridine, sulfolane, tetrahydrofuran (THF), tetralin, toluene, 1,1,2-trichloroethene and xylene, acetic acid, acetone Anisole, 1-butanol, 2-butanol, butyl acetate, tert-butyl methyl ether, cumene, heptane, isobutyl acetate, isopropyl acetate, methyl acetate, 3-methyl-1-butanol, methyl ethyl Selected from ketone, methyl isobutyl ketone, 2-methyl-1-propanol, ethyl acetate, ethyl ether, ethyl formate, pentane, 1-pentanol, 1-propanol, 2-propanol, propyl acetate or any combination thereof 51. The method of claim 50, further comprising one or more organic solvents. The solvent system is chlorobenzene, cyclohexane, 1,2-dichloroethane, dichloromethane, 1,2-dimethoxyethane, formamide, hexane, 2-methoxyethanol, methylbutylketone, methylcyclohexane, nitromethane, tetralin, xylene, toluene, 1, 1,2-trichloroethane, acetone, anisole, 1-butanol, 2-butanol, butyl acetate, tert-butyl methyl ether, cumene, ethanol, ethyl acetate, ethyl ether, ethyl formate, heptane, isobutyl acetate, isopropyl acetate, methyl acetate , 3-methyl-1-butanol, methyl ethyl ketone, 2-methyl-1-propanol, pentane, 1-propanol, 1-pentanol, 2-propanol, propyl acetate, tetrahydrofuran Other further comprises one or more organic solvents selected from methyl tetrahydrofuran The method of claim 51. The solvent system is 2-ethoxyethanol, ethylene glycol, methanol, 2-methoxyethanol, 1-butanol, 2-butanol, 3-methyl-1-butanol, 2-methyl-1-propanol, ethanol, 1-pentanol. 1-propanol, 2-propanol, methyl butyl ketone, acetone, methyl ethyl ketone, methyl isobutyl ketone, butyl acetate, isobutyl acetate, isopropyl acetate, methyl acetate, ethyl acetate, propyl acetate, pyridine, toluene, xylene or any combination thereof 52. The method of claim 51, further comprising one or more organic solvents selected from. 52. The method of claim 51, wherein the solvent system further comprises one or more organic solvents selected from isopropanol, acetone, or any combination thereof. A method for preparing an HCl salt of Form D of Compound (1), wherein Compound (1) has the following structural formula:

The method is represented by
A method comprising dehydrating HCl salt of Compound (1) Form 1 / 2H ₂ O in Form A. A process for preparing Form A compound (1), wherein compound (1) has the following structural formula:

The method is represented by
(A) A method comprising stirring an amorphous compound (1) or a solvate of the compound (1) in a solvent system containing water and ethanol. 57. The method of claim 56, wherein the stirring step is performed at a temperature within the range of 18 [deg.] C to 90 [deg.] C. 58. The method of any of claims 56 or 57, wherein the solvent system comprises 5 wt% to 15 wt% water based on the weight of the solvent system. (B) stirring the amorphous compound (1) in nitromethane to form a seed crystal of the A form compound (1); and (c) a seed crystal of the A form compound (1). 59. The method according to any one of claims 56 to 58, further comprising adding to the mixture obtained in the mixing step (a). 60. A method according to any one of claims 56 to 59, wherein the stirring step (a) is performed at the reflux temperature of the solvent system. (B) a step of forming a seed crystal of the compound (1) in form A by stirring the amorphous compound (1) in nitromethane; and (c) the mixture obtained in the mixing step (a). Cooling to a temperature in the range of 18 ° C. to 60 ° C .; and (d) adding seed crystals of the Form A compound (1) to the mixture obtained in step (c). The method according to any one of 56 to 58. After the addition of water, a sufficient amount of water is added so that the resulting solvent system will contain 15 wt% to 25 wt% of water before the seed crystals of Form A compound (1) are added. 62. The method of claim 61, further comprising adding to the mixture obtained via the process. After adding water, adding a sufficient amount of water to the mixture containing seed crystals of Form A compound (1), such that the resulting solvent system will contain 35 wt% to 45 wt% water. 62. The method of claim 61, comprising. 62. The method of claim 61, further comprising cooling the mixture comprising seed crystals of Form A compound (1) to a temperature between 0 <0> C and 10 <0> C after the addition of water. A method for preparing a tosylate salt of Form A compound (1), wherein compound (1) has the following structural formula:

Wherein the method comprises stirring a mixture of a solvent system comprising amorphous compound (1) or a solvate of compound (1), p-toluenesulfonic acid, and acetonitrile. 2-methyltetrahydrofuran solvate of compound (1), wherein compound (1) has the following structural formula:

2-Methyltetrahydrofuran solvate of compound (1) represented by: 25. An administration regimen comprising the step of administering to the subject a polymorphic compound (1) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 24 in a dose of 100 mg to 1,600 mg. Wherein the dosage is administered once, twice or three times per day. 68. The dosage regimen of claim 67, wherein the dosage is from 300 mg to 1,600 mg. 69. A dosage regimen according to claim 68, wherein the dosage is between 600 mg and 1200 mg. 70. The dosage regimen of claim 69, wherein the dosage is administered once per day. 71. A dosage regimen according to claim 70, wherein the dosage is 600 mg or 800 mg. 69. A dosage regimen according to claim 68, wherein the dosage is from 300 mg to 900 mg. 73. A dosage regimen according to claim 72, wherein the dosage is administered twice per day. 69. A dosage regimen according to claim 68, wherein the dosage is 400 mg or 600 mg. 75. Dosage regimen according to any one of claims 67 to 74, wherein the polymorphic compound (1) or a pharmaceutically acceptable salt thereof is administered over a treatment period from one day to the entire influenza season. 76. A dosage regimen according to claim 75, wherein the treatment period is from 3 days to 14 days. 77. A dosage regimen according to claim 76, wherein the treatment period is 3 days, 4 days or 5 days. A loading dose of 600 mg to 1,600 mg is administered to the subject on day 1 and a dosage of 400 mg to 1,200 mg is administered to the subject over the remainder of the treatment period. 78. A dosage regimen according to any one of 67-77. A loading dose of 900 mg to 1,600 mg is administered to the subject on day 1 and a dosage of 400 mg to 1,200 mg is administered to the subject over the remainder of the treatment period. 78. Administration regimen according to 78. 80. A loading dose of 900 mg or 1,200 mg is administered to the subject on day 1 and a dosage of 600 mg to 800 mg is administered to the subject over the remainder of the treatment period. The administration regime described. 81. The loading dose of 900 mg is administered to the subject on day 1 and the 600 mg dose is administered to the subject once a day for the remainder of the treatment period. Dosing regimen. 81. A loading dose of 1,200 mg is administered to the subject on day 1 and a dosage of 600 mg is administered to the subject once a day for the remainder of the treatment period. The administration regime described.

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